Cln101 antibody compositions and methods of use alone and in combination with prostate specific antigen and other cancer markers

ABSTRACT

This invention relates to a method for assessing risk of prostate and/or ovarian cancer. Specifically, in one embodiment it relates to utilizing both Cln101 and Prostate Specific Antigen (PSA) in combination to determine the risk of prostate cancer. In an alternative embodiment, the invention is relates to utilizing Cln101 alone or in combination with CA125 to determine the risk of ovarian cancer. The invention further provides isolated anti-prostate or ovarian cancer antigen (Cln101) antibodies that bind to Cln101 in vivo. The invention also encompasses compositions comprising an anti-Cln101 antibody and a carrier. These compositions can be provided in an article of manufacture or a kit. Another aspect of the invention is an isolated nucleic acid encoding an anti-Cln101 antibody, as well as an expression vector comprising the isolated nucleic acid. Also provided are cells that produce the anti-Cln101 antibodies. The invention encompasses a method of producing the anti-Cln10I antibodies. Other aspects of the invention are a method of killing an Cln101-expressing cancer cell, comprising contacting the cancer cell with an anti-Cln101 antibody and a method of alleviating or treating an Cln101-expressing cancer in a mammal, comprising administering a therapeutically effective amount of the anti-Cln101 antibody to the mammal.

This patent application claims the benefit of priority to U.S.Provisional Patent Application Ser. No. 60/556,239, filed Mar. 25, 2004and U.S. Provisional Patent Application Ser. No. 60/474,110, filed May29, 2003, each of which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to anti-Cln101 antibody compositions andmethods for assessing risk of prostate and/or ovarian cancer.Specifically, it relates to utilizing both Cln101 (also known asRegenerating Protein IV or Reg IV) and Prostate Specific Antigen (PSA)in combination to detect prostate cancer. In addition, this invention isrelated to the use of Cln101 alone or in combination to detect ovariancancer.

BACKGROUND OF THE INVENTION

Prostate Cancer

Prostate cancer is the most prevalent cancer in men and is the secondleading cause of death from cancer among males in the United States.AJCC Cancer Staging Handbook 203 (Irvin D. Fleming et al. eds., 5^(th)ed. 1998); Walter J. Burdette, Cancer: Etiology, Diagnosis, andTreatment 147 (1998). In 1999, it was estimated that 37,000 men in theUnited States would die as result of prostate cancer. Elizabeth A. Platzet al., & Edward Giovannucci, Epidemiology of and Risk Factors forProstate Cancer, in Management of Prostate Cancer 21 (Eric A Klein, ed.2000). More recently, the American Cancer Society estimated there willbe 230,110 new cases of prostate cancer and 29,900 deaths in 2004.American Cancer Society website: cancer with the extension org of theworld wide web. Cancer of the prostate typically occurs in older males,with a median age of 74 years for clinical diagnosis. Burdette, supra at147. A man's risk of being diagnosed with invasive prostate cancer inhis lifetime is one in six. Platz et al., supra at 21.

Although our understanding of the etiology of prostate cancer isincomplete, the results of extensive research in this area point to acombination of age, genetic and environmental/dietary factors. Platz etal., supra at 19; Burdette, supra at 147; Steven K. Clinton, Diet andNutrition in Prostate Cancer Prevention and Therapy, in Prostate Cancer:a Multidisciplinary Guide 246-269 (Philip W. Kantoff et al. eds. 1997).Broadly speaking, genetic risk factors predisposing one to prostatecancer include race and a family history of the disease. Platz et al.,supra at 19, 28-29, 32-34. Aside from these generalities, a deeperunderstanding of the genetic basis of prostate cancer has remainedelusive. Considerable research has been directed to studying the linkbetween prostate cancer, androgens, and androgen regulation, asandrogens play a crucial role in prostate growth and differentiation.Meena Augustus et al., Molecular Genetics and Markers of Progression, inManagement of Prostate Cancer 59 (Eric A Klein ed. 2000). While a numberof studies have concluded that prostate tumor development is linked toelevated levels of circulating androgen (e.g., testosterone anddihydrotestosterone), the genetic determinants of these levels remainunknown. Platz et al., supra at 29-30.

Several studies have explored a possible link between prostate cancerand the androgen receptor (AR) gene, the gene product of which mediatesthe molecular and cellular effects of testosterone anddihydrotestosterone in tissues responsive to androgens. Id. at 30.Differences in the number of certain trinucleotide repeats in exon 1,the region involved in transactivational control, have been ofparticular interest. Augustus et al., supra at 60. For example, thesestudies have revealed that as the number of CAG repeats decreases thetransactivation ability of the gene product increases, as does the riskof prostate cancer. Platz et al., supra at 30-31. Other research hasfocused on the α-reductase Type 2 gene, the gene which codes for theenzyme that converts testosterone into dihydrotestosterone. Id. at 30.Dihydrotestosterone has greater affinity for the AR than testosterone,resulting in increased transactivation of genes responsive to androgens.Id. While studies have reported differences among the races in thelength of a TA dinucleotide repeat in the 3′ untranslated region, nolink has been established between the length of that repeat and prostatecancer. Id. Interestingly, while ras gene mutations are implicated innumerous other cancers, such mutations appear not to play a significantrole in prostate cancer, at least among Caucasian males. Augustus, supraat 52.

Environmental/dietary risk factors which may increase the risk ofprostate cancer include intake of saturated fat and calcium. Platz etal., supra at 19, 25-26. Conversely, intake of selenium, vitamin E andtomato products (which contain the carotenoid lycopene) apparentlydecrease that risk. Id. at 19, 26-28 The impact of physical activity,cigarette smoking, and alcohol consumption on prostate cancer isunclear. Platz et al., supra at 23-25.

Periodic screening for prostate cancer is most effectively performed bydigital rectal examination (DRE) of the prostate, in conjunction withdetermination of the serum level of prostate-specific antigen (PSA).Burdette, supra at 148. While the merits of such screening are thesubject of considerable debate, Jerome P. Richie & Irving D. Kaplan,Screening for Prostate Cancer: The Horns of a Dilemma, in ProstateCancer: A Multidisciplinary Guide 1-10 (Philip W. Kantoff et al. eds.1997), the American Cancer Society and American Urological Associationrecommend that both of these tests be performed annually on men 50 yearsor older with a life expectancy of at least 10 years, and younger men athigh risk for prostate cancer. Ian M. Thompson & John Foley, Screeningfor Prostate Cancer, in Management of Prostate Cancer 71 (Eric A Kleined. 2000). If necessary, these screening methods may be followed byadditional tests, including biopsy, ultrasonic imaging, computerizedtomography, and magnetic resonance imaging. Christopher A. Haas & MartinI. Resnick, Trends in Diagnosis, Biopsy, and Imaging, in Management ofProstate Cancer 89-98 (Eric A Klein ed. 2000); Burdette, supra at 148.

Once the diagnosis of prostate cancer has been made, treatment decisionsfor the individual are typically linked to the stage of prostate cancerpresent in that individual, as well as his age and overall health.Burdette, supra at 151. One preferred classification system for stagingprostate cancer was developed by the American Urological Association(AUA). Id. at 148. The AUA classification system divides prostate tumorsinto four broad stages, A to D, which are in turn accompanied by anumber of smaller substages. Burdette, supra at 152-153; Anthony V.D'Amico et al., The Staging of Prostate Cancer, in Prostate Cancer: AMultidisciplinary Guide 41 (Philip W. Kantoff et al. eds. 1997).

Stage A prostate cancer refers to the presence of microscopic cancerwithin the prostate gland. D'Amico, supra at 41. This stage is comprisedof two substages: A1, which involves less than four well-differentiatedcancer foci within the prostate, and A2, which involves greater thanthree well-differentiated cancer foci or alternatively, moderately topoorly differentiated foci within the prostate. Burdette, supra at 152;D'Amico, supra at 41. Treatment for stage A1 preferentially involvesfollowing PSA levels and periodic DRE. Burdette, supra at 151. ShouldPSA levels rise, preferred treatments include radical prostatectomy inpatients 70 years of age and younger, external beam radiotherapy forpatients between 70 and 80 years of age, and hormone therapy for thoseover 80 years of age. Id.

Stage B prostate cancer is characterized by the presence of a palpablelump within the prostate. Burdette, supra at 152-53; D'Amico, supra at41. This stage is comprised of three substages: B1, in which the lump isless than 2 cm and is contained in one lobe of the prostate; B2, inwhich the lump is greater than 2 cm yet is still contained within onelobe; and B3, in which the lump has spread to both lobes. Burdette,supra, at 152-53. For stages B1 and B2, the treatment again involvesradical prostatectomy in patients 70 years of age and younger, externalbeam radiotherapy for patients between 70 and 80 years of age, andhormone therapy for those over 80 years of age. Id. at 151. In stage B3,radical prostatectomy is employed if the cancer is well-differentiatedand PSA levels are below 15 ng/mL; otherwise, external beam radiation isthe chosen treatment option. Id.

Stage C prostate cancer involves a substantial cancer mass accompaniedby extraprostatic extension. Burdette, supra at 153; D'Amico, supra at41. Like stage A prostate cancer, Stage C is comprised of two substages:substage C1, in which the tumor is relatively minimal, with minorprostatic extension, and substage C2, in which the tumor is large andbulky, with major prostatic extension. Id. The treatment of choice forboth substages is external beam radiation. Burdette, supra at 151.

The fourth and final stage of prostate cancer, Stage D, describes theextent to which the cancer has metastasized. Burdette, supra at 153;D'Amico, supra at 41. This stage is comprised of four substages: (1) D0,in which acid phophatase levels are persistently high, (2) D1, in whichonly the pelvic lymph nodes have been invaded, (3) D2, in which thelymph nodes above the aortic bifurcation have been invaded, with orwithout distant metastasis, and (4) D3, in which the metastasisprogresses despite intense hormonal therapy. Id. Treatment at this stagemay involve hormonal therapy, chemotherapy, and removal of one or bothtestes. Burdette, supra at 151.

Despite the need for accurate staging of prostate cancer, currentstaging methodology is limited. The wide variety of biological behaviordisplayed by neoplasms of the prostate has resulted in considerabledifficulty in predicting and assessing the course of prostate cancer.Augustus et al., supra at 47. Indeed, despite the fact that mostprostate cancer patients have carcinomas that are of intermediate gradeand stage, prognosis for these types of carcinomas is highly variable.Andrew A Renshaw & Christopher L. Corless, Prognostic Features in thePathology of Prostate Cancer, in Prostate Cancer: A MultidisciplinaryGuide 26 (Philip W. Kantoff et al. eds. 1997). Techniques such astransrectal ultrasound, abdominal and pelvic computerized tomography,and MRI have not been particularly useful in predicting local tumorextension. D'Amico, supra at 53 (editors' comment). While the use ofserum PSA in combination with the Gleason score is currently the mosteffective method of staging prostate cancer, id., PSA is of limitedpredictive value, Augustus et al., supra at 47; Renshaw et al., supra at26, and the Gleason score is prone to variability and error, King, C. R.& Long, J. P., Int'l. J Cancer 90(6): 326-30 (2000). As such, thecurrent focus of prostate cancer research has been to obtain biomarkersto help better assess the progression of the disease. Augustus et al.,supra at 47; Renshaw et al., supra at 26; Pettaway, C. A., Tech. Urol.4(1): 35-42 (1998).

Current Therapeutics

Radical Prostatectomy

This operation removes the entire prostate gland plus some tissue aroundit and is used most often if the cancer is thought not to have spreadoutside of the gland. There are two main types of radical prostatectomy:radical retropubic prostatectomy and radical perineal prostatectomy. Inthe retropubic operation, the surgeon makes a skin incision in the lowerabdomen. The surgeon can remove lymph nodes during this operationthrough the same incision. A nerve-sparing radical retropubicprostatectomy is a modification of this operation.

The radical peritoneal prostatectomy removes the prostate through anincision in the skin between the scrotum and anus. Nerve-sparingoperations are more difficult by this approach and lymph nodes cannot beremoved through this incision. If lymph node examination is needed formen having a radical peritoneal prostatectomy, the surgeon can removesome lymph nodes through a very small skin incision in the abdomen or byusing a laparoscope. A laparoscope is a long slender tube through whicha surgeon can view and remove lymph nodes near the prostate gland.

Radiation Therapy

Radiation is sometimes used to treat prostate cancer that is stillconfined to the prostate gland, or has spread to nearby tissue. If thedisease is more advanced, radiation may be used to reduce the size ofthe tumor. The two main types of radiation therapy are external beamradiation and brachytherapy (internal radiation) and internal radiationtherapy (brachytherapy). Internal radiation therapy uses smallradioactive pellets (each about the size of a grain of rice) that aredirectly implanted (permanently or temporarily) into the prostate.

Hormone Therapy

This treatment is often used for patients whose prostate cancer hasspread beyond the prostate or has recurred after treatment. The goal ofhormone therapy is to lower levels of the male hormones, androgens. Themain androgen is called testosterone. Androgens are produced mainly inthe testicles and cause prostate cancer cells to grow. Lowering androgenlevels can make prostate cancers shrink or grow more slowly. But,hormone therapy does not cure the cancer. There are several methods usedfor hormone therapy.

Some prostate cancers do not respond to hormone therapy, and are calledandrogen independent cancers. Some prostate cancers respond to hormonaltherapy for a few years before becoming androgen independent. Lessoften, prostate cancers may be androgen independent at the time they arediagnosed.

Orchiectomy: This operation removes the testicles. Although it is asurgical treatment, orchiectomy is considered hormonal therapy becauseit works by removing the main source of male hormones.

Luteinizing hormone-releasing hormone (LHRH) analogs: These drugs candecrease the amount of testosterone produced by a man's testicles, aseffectively as surgical removal of the testicles. LHRH analogs (alsocalled LHRH agonists) are injected either monthly or every three months.The two LHRH analogs currently available in the United States areleuprolide (Lupron®) and goserelin (Zoladex®).

Anti-androgens: Even after orchiectomy or during treatment with LHRHanalogs, a small amount of androgen is still produced by the adrenalglands. Anti-androgens block the body's ability to use androgens. Drugsof this type, such as flutamide (Eulexin®), bicalutamide (Casodex®), andnilutamide (Nilandron®), are taken as pills, once or three times a day.Anti-androgens are often used in combination with orchiectomy or LHRHanalogs. This combination is called total androgen blockade.

Other hormonal drugs: Megestrol acetate (Megace®) andmedroxyprogesterone (Depo-Provera®) are sometimes used if “first-line”hormonal treatments lose effectiveness. Ketoconazole (Nizoral®),initially used for treating fungal infections and later found to alsowork as an anti-androgen, is another drug for “second line” hormonaltherapy.

Chemotherapy

Chemotherapy is an option for patients whose prostate cancer has spreadoutside of the prostate gland and for whom hormone therapy has failed.It is not expected to destroy all of the cancer cells, but it may slowtumor growth and reduce pain.

Some of the chemotherapy drugs used in treating prostate cancer that hasreturned or continued to grow and spread after treatment with hormonaltherapy include doxorubicin (Adriamycin), estramustine, etoposide,mitoxantrone, vinblastine, and paclitaxel. Two or more drugs are oftengiven together to reduce the likelihood of the cancer cells becomingresistant to chemotherapy. Small cell carcinoma is a rare type ofprostate cancer that is more likely to respond to chemotherapy than tohormonal therapy. Small cell carcinoma develops more often in the lungsthan in the prostate. Since small cell lung cancer often responds tochemotherapy with cisplatin and etoposide, these drugs are recommendedfor treating small cell cancers that develop in the prostate.

Accordingly, there is a great need for more sensitive and accuratemethods for predicting whether a person is likely to develop prostatecancer, for diagnosing prostate cancer, for monitoring the progressionof the disease, for staging the prostate cancer, for determining whetherthe prostate cancer has metastasized and for imaging the prostatecancer. There is also a need for better treatment of prostate cancer.

Ovarian Cancer

Cancer of the ovaries is the fourth-most common cause of cancer death inwomen in the United States, with more than 23,000 new cases and roughly14,000 deaths predicted for the year 2001. Shridhar, V. et al., CancerRes. 61(15): 5895-904 (2001); Memarzadeh, S. & Berek, J. S., J. Reprod.Med. 46(7): 621-29 (2001). The American Cancer Society estimates thatthere will be about 25,580 new cases of ovarian cancer in 2004 in theUnited States alone. Ovarian cancer will cause about 16,090 deaths inthe United States in the same year. ACS Website: cancer with theextension org of the world wide web. The incidence of ovarian cancer isof serious concern worldwide, with an estimated 191,000 new casespredicted annually. Runnebaum, I. B. & Stickeler, E., J. Cancer Res.Clin. Oncol. 127(2): 73-79 (2001). Unfortunately, women with ovariancancer are typically asymptomatic until the disease has metastasized.Because effective screening for ovarian cancer is not available, roughly70% of women diagnosed have an advanced stage of the cancer with afive-year survival rate of 25-30%. Memarzadeh, S. & Berek, J. S., supra;Nunns, D. et al., Obstet. Gynecol. Surv. 55(12): 746-51. Conversely,women diagnosed with early stage ovarian cancer enjoy considerablyhigher survival rates. Werness, B. A. & Eltabbakh, G. H., Int'l. J.Gynecol. Pathol. 20(1): 48-63 (2001). Although our understanding of theetiology of ovarian cancer is incomplete, the results of extensiveresearch in this area point to a combination of age, genetics,reproductive, and dietary/environmental factors. Age is a key riskfactor in the development of ovarian cancer: while the risk fordeveloping ovarian cancer before the age of 30 is slim, the incidence ofovarian cancer rises linearly between ages 30 to 50, increasing at aslower rate thereafter, with the highest incidence being amongseptuagenarian women. Jeanne M. Schilder et al., Hereditary OvarianCancer: Clinical Syndromes and Management, in Ovarian Cancer 182(Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001).

With respect to genetic factors, a family history of ovarian cancer isthe most significant risk factor in the development of the disease, withthat risk depending on the number of affected family members, the degreeof their relationship to the woman, and which particular first degreerelatives are affected by the disease. Id. Mutations in several geneshave been associated with ovarian cancer, including BRCA1 and BRCA2,both of which play a key role in the development of breast cancer, aswell as hMSH2 and hMLH1, both of which are associated with hereditarynon-polyposis colon cancer. Katherine Y. Look, Epidemiology, Etiology,and Screening of Ovarian Cancer, in Ovarian Cancer 169, 171-73 (StephenC. Rubin & Gregory P. Sutton eds., 2d ed. 2001). BRCA1, located onchromosome 17, and BRCA2, located on chromosome 13, are tumor suppressorgenes implicated in DNA repair; mutations in these genes are linked toroughly 10% of ovarian cancers. Id. at 171-72; Schilder et al., supra at185-86. hMSH2 and hMLH1 are associated with DNA mismatch repair, and arelocated on chromosomes 2 and 3, respectively; it has been reported thatroughly 3% of hereditary ovarian carcinomas are due to mutations inthese genes. Look, supra at 173; Schilder et al., supra at 184, 188-89.

Reproductive factors have also been associated with an increased orreduced risk of ovarian cancer. Late menopause, nulliparity, and earlyage at menarche have all been linked with an elevated risk of ovariancancer. Schilder et al., supra at 182. One theory hypothesizes thatthese factors increase the number of ovulatory cycles over the course ofa woman's life, leading to “incessant ovulation,” which is thought to bethe primary cause of mutations to the ovarian epithelium. Id.; Laura J.Havrilesky & Andrew Berchuck, Molecular Alterations in Sporadic OvarianCancer, in Ovarian Cancer 25 (Stephen C. Rubin & Gregory P. Sutton eds.,2d ed. 2001). The mutations may be explained by the fact that ovulationresults in the destruction and repair of that epithelium, necessitatingincreased cell division, thereby increasing the possibility that anundetected mutation will occur. Id. Support for this theory may be foundin the fact pregnancy, lactation, and the use of oral contraceptives,all of which suppress ovulation, confer a protective effect with respectto developing ovarian cancer. Id.

Among dietary/environmental factors, there would appear to be anassociation between high intake of animal fat or red meat and ovariancancer, while the antioxidant Vitamin A, which prevents free radicalformation and also assists in maintaining normal cellulardifferentiation, may offer a protective effect. Look, supra at 169.Reports have also associated asbestos and hydrous magnesium trisilicate(talc), the latter of which may be present in diaphragms and sanitarynapkins. Id. at 169-70.

Current screening procedures for ovarian cancer, while of some utility,are quite limited in their diagnostic ability, a problem that isparticularly acute at early stages of cancer progression when thedisease is typically asymptomatic yet is most readily treated. Walter J.Burdette, Cancer: Etiology, Diagnosis, and Treatment 166 (1998);Memarzadeh & Berek, supra; Runnebaum & Stickeler, supra; Werness &Eltabbakh, supra. Commonly used screening tests include biannualrectovaginal pelvic examination, radioimmunoassay to detect the CA-125serum tumor marker, and transvaginal ultrasonography. Burdette, supra at166.

Pelvic examination has failed to yield adequate numbers of earlydiagnoses, and the other methods are not sufficiently accurate. Id. Onestudy reported that only 15% of patients who suffered from ovariancancer were diagnosed with the disease at the time of their pelvicexamination. Look, supra at 174. Moreover, the CA-125 test is prone togiving false positives in pre-menopausal women and has been reported tobe of low predictive value in post-menopausal women. Id. at 174-75.Although transvaginal ultrasonography is now the preferred procedure forscreening for ovarian cancer, it is unable to distinguish reliablybetween benign and malignant tumors, and also cannot locate primaryperitoneal malignancies or ovarian cancer if the ovary size is normal.Schilder et al., supra at 194-95. While genetic testing for mutations ofthe BRCA1, BRCA2, hMSH2, and hMLH1 genes is now available, these testsmay be too costly for some patients and may also yield false negative orindeterminate results. Schilder et al., supra at 191-94.

Other markers of interest are HE4 and mesothelin, see Urban et al.Ovarian cancer screening Hematol Oncol Clin North Am. 2003 August;17(4):989-1005; Hellstrom et al. The HE4 (WFDC2) protein is a biomarkerfor ovarian carcinoma, Cancer Res. 2003 Jul. 1; 63(13):3695-700;Ordonez, Application of mesothelin immunostaining in tumor diagnosis, AmJ Surg Pathol. 2003 November; 27(11):1418-28.

The staging of ovarian cancer, which is accomplished through surgicalexploration, is crucial in determining the course of treatment andmanagement of the disease. AJCC Cancer Staging Handbook 187 (Irvin D.Fleming et al. eds., 5th ed. 1998); Burdette, supra at 170; Memarzadeh &Berek, supra; Shridhar et al., supra. Staging is performed by referenceto the classification system developed by the International Federationof Gynecology and Obstetrics. David H. Moore, Primary SurgicalManagement of Early Epithelial Ovarian Carcinoma, in Ovarian Cancer 203(Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001); Fleming et al.eds., supra at 188. Stage I ovarian cancer is characterized by tumorgrowth that is limited to the ovaries and is comprised of threesubstages. Id. In substage IA, tumor growth is limited to one ovary,there is no tumor on the external surface of the ovary, the ovariancapsule is intact, and no malignant cells are present in ascites orperitoneal washings. Id. Substage IB is identical to A1, except thattumor growth is limited to both ovaries. Id. Substage IC refers to thepresence of tumor growth limited to one or both ovaries, and alsoincludes one or more of the following characteristics: capsule rupture,tumor growth on the surface of one or both ovaries, and malignant cellspresent in ascites or peritoneal washings. Id.

Stage II ovarian cancer refers to tumor growth involving one or bothovaries, along with pelvic extension. Id. Substage IIA involvesextension and/or implants on the uterus and/or fallopian tubes, with nomalignant cells in the ascites or peritoneal washings, while substageIIB involves extension into other pelvic organs and tissues, again withno malignant cells in the ascites or peritoneal washings. Id. SubstageIIC involves pelvic extension as in IIA or IIB, but with malignant cellsin the ascites or peritoneal washings. Id.

Stage III ovarian cancer involves tumor growth in one or both ovaries,with peritoneal metastasis beyond the pelvis confirmed by microscopeand/or metastasis in the regional lymph nodes. Id. Substage IIIA ischaracterized by microscopic peritoneal metastasis outside the pelvis,with substage IIIB involving macroscopic peritoneal metastasis outsidethe pelvis 2 cm or less in greatest dimension. Id. Substage IIIC isidentical to IIIB, except that the metastasis is greater than 2 cm ingreatest dimension and may include regional lymph node metastasis. Id.Lastly, Stage IV refers to the presence distant metastasis, excludingperitoneal metastasis. Id.

While surgical staging is currently the benchmark for assessing themanagement and treatment of ovarian cancer, it suffers from considerabledrawbacks, including the invasiveness of the procedure, the potentialfor complications, as well as the potential for inaccuracy. Moore, supraat 206-208, 213. In view of these limitations, attention has turned todeveloping alternative staging methodologies through understandingdifferential gene expression in various stages of ovarian cancer and byobtaining various biomarkers to help better assess the progression ofthe disease. Vartiainen, J. et al., Int'l J. Cancer, 95(5): 313-16(2001); Shridhar et al. supra; Baekelandt, M. et al., J. Clin. Oncol.18(22): 3775-81.

The treatment of ovarian cancer typically involves a multiprong attack,with surgical intervention serving as the foundation of treatment.Dennis S. Chi & William J. Hoskins, Primary Surgical Management ofAdvanced Epithelial Ovarian Cancer, in Ovarian Cancer 241 (Stephen C.Rubin & Gregory P. Sutton eds., 2d ed. 2001). For example, in the caseof epithelial ovarian cancer, which accounts for ˜90% of cases ofovarian cancer, treatment typically consists of: (1) cytoreductivesurgery, including total abdominal hysterectomy, bilateralsalpingo-oophorectomy, omentectomy, and lymphadenectomy, followed by (2)adjuvant chemotherapy with paclitaxel and either cisplatin orcarboplatin. Eltabbakh, G. H. & Awtrey, C. S., Expert Op. Pharmacother.2(10): 109-24. Despite a clinical response rate of 80% to the adjuvanttherapy, most patients experience tumor recurrence within three years oftreatment. Id. Certain patients may undergo a second cytoreductivesurgery and/or second-line chemotherapy. Memarzadeh & Berek, supra.

From the foregoing, it is clear that procedures used for detecting,diagnosing, monitoring, staging, prognosticating, and preventing therecurrence of ovarian cancer are of critical importance to the outcomeof the patient. Moreover, current procedures, while helpful in each ofthese analyses, are limited by their specificity, sensitivity,invasiveness, and/or their cost. As such, highly specific and sensitiveprocedures that would operate by way of detecting novel markers incells, tissues, or bodily fluids, with minimal invasiveness and at areasonable cost, would be highly desirable.

Accordingly, there is a great need for more sensitive and accuratemethods for predicting whether a person is likely to develop ovariancancer, for diagnosing ovarian cancer, for monitoring the progression ofthe disease, for staging the ovarian cancer, for determining whether theovarian cancer has metastasized, for imaging the ovarian cancer and forbetter treatment of ovarian cancer.

Angiogenesis in Cancer

Growth and metastasis of solid tumors are also dependent onangiogenesis. Folkman, J., 1986, Cancer Research, 46, 467-473; Folkman,J., 1989, Journal of the National Cancer Institute, 82, 4-6. It has beenshown, for example, that tumors which enlarge to greater than 2 mm mustobtain their own blood supply and do so by inducing the growth of newcapillary blood vessels. Once these new blood vessels become embedded inthe tumor, they provide a means for tumor cells to enter the circulationand metastasize to distant sites such as liver, lung or bone. Weidner,N., et al., 1991, The New England Journal of Medicine, 324(1), 1-8.

Angiogenesis, defined as the growth or sprouting of new blood vesselsfrom existing vessels, is a complex process that primarily occurs duringembryonic development. The process is distinct from vasculogenesis, inthat the new endothelial cells lining the vessel arise fromproliferation of existing cells, rather than differentiating from stemcells. The process is invasive and dependent upon proteolysis of theextracellular matrix (ECM), migration of new endothelial cells, andsynthesis of new matrix components. Angiogenesis occurs duringembryogenic development of the circulatory system; however, in adulthumans, angiogenesis only occurs as a response to a pathologicalcondition (except during the reproductive cycle in women).

Under normal physiological conditions in adults, angiogenesis takesplace only in very restricted situations such as hair growth andwounding healing. Auerbach, W. and Auerbach, R., 1994, Pharmacol Ther.63(3):265-3 11; Ribatti et al., 1991, Haematologica 76(4):3 11-20;Risau, 1997, Nature 386(6626):67 14. Angiogenesis progresses by astimulus which results in the formation of a migrating column ofendothelial cells. Proteolytic activity is focused at the advancing tipof this “vascular sprout”, which breaks down the ECM sufficiently topermit the column of cells to infiltrate and migrate. Behind theadvancing front, the endothelial cells differentiate and begin to adhereto each other, thus forming a new basement membrane. The cells thencease proliferation and finally define a lumen for the new arteriole orcapillary.

Unregulated angiogenesis has gradually been recognized to be responsiblefor a wide range of disorders, including, but not limited to, cancer,cardiovascular disease, rheumatoid arthritis, psoriasis and diabeticretinopathy. Folkman, 1995, Nat Med 1(1):27-31; Isner, 1999, Circulation99(13): 1653-5; Koch, 1998, Arthritis Rheum 41(6):951-62; Walsh, 1999,Rheumatology (Oxford) 38(2): 103-12; Ware and Simons, 1997, Nat Med3(2): 158-64.

Of particular interest is the observation that angiogenesis is requiredby solid tumors for their growth and metastases. Folkman, 1986 supra;Folkman 1990, J Natl. Cancer Inst., 82(1) 4-6; Folkman, 1992, SeminCancer Biol 3(2):65-71; Zetter, 1998, Annu Rev Med 49:407-24. A tumorusually begins as a single aberrant cell which can proliferate only to asize of a few cubic millimeters due to the distance from availablecapillary beds, and it can stay ‘dormant’ without further growth anddissemination for a long period of time. Some tumor cells then switch tothe angiogenic phenotype to activate endothelial cells, whichproliferate and mature into new capillary blood vessels. These newlyformed blood vessels not only allow for continued growth of the primarytumor, but also for the dissemination and recolonization of metastatictumor cells. The precise mechanisms that control the angiogenic switchis not well understood, but it is believed that neovascularization oftumor mass results from the net balance of a multitude of angiogenesisstimulators and inhibitors Folkman, 1995, supra.

One of the most potent angiogenesis inhibitors is endostatin identifiedby O'Reilly and Folkman. O'Reilly et al., 1997, Cell 88(2):277-85;O'Reilly et al., 1994, Cell 79(2):3 15-28. Its discovery was based onthe phenomenon that certain primary tumors can inhibit the growth ofdistant metastases. O'Reilly and Folkman hypothesized that a primarytumor initiates angiogenesis by generating angiogenic stimulators inexcess of inhibitors. However, angiogenic inhibitors, by virtue of theirlonger half life in the circulation, reach the site of a secondary tumorin excess of the stimulators. The net result is the growth of primarytumor and inhibition of secondary tumor. Endostatin is one of a growinglist of such angiogenesis inhibitors produced by primary tumors. It is aproteolytic fragment of a larger protein: endostatin is a 20 kDafragment of collagen XVIII (amino acid H1132-K1315 in murine collagenXVIII). Endostatin has been shown to specifically inhibit endothelialcell proliferation in vitro and block angiogenesis in vivo. Moreimportantly, administration of endostatin to tumor-bearing mice leads tosignificant tumor regression, and no toxicity or drug resistance hasbeen observed even after multiple treatment cycles. Boehm et al., 1997,Nature 390(6658):404-407. The fact that endostatin targets geneticallystable endothelial cells and inhibits a variety of solid tumors makes ita very attractive candidate for anticancer therapy. Fidler and Ellis,1994, Cell 79(2): 185-8; Gastl et al., 1997, Oncology 54(3):177-84;Hinsbergh et al., 1999, Ann Oncol 10 Suppl 4:60-3. In addition,angiogenesis inhibitors have been shown to be more effective whencombined with radiation and chemotherapeutic agents. Klement, 2000, J.Clin Invest, 105(8) R15-24. Browder, 2000, Cancer Res. 6-(7) 1878-86,Arap et al., 1998, Science 279(5349):377-80; Mauceri et al., 1998,Nature 394(6690):287-91.

The present invention provides alternative methods of detecting prostateand ovarian cancer that overcome the limitations of conventionaldiagnostic methods as well as offer additional advantages that will beapparent from the detailed description below. Furthermore, the presentinvention provides alternative methods of treating prostate and ovariancancer that overcome the limitations of conventional therapeutic methodsas well as offer additional advantages that will be apparent from thedetailed description below.

SUMMARY OF THE INVENTION

This invention is directed to a method for assessing risk of prostatecancer in a patient which comprises measuring levels of both Cln101 andProstate Specific Antigen (PSA) in the patient, analyzing a riskassociated with the level of PSA and a risk associated with the level ofCln101, and using the combined risks to assess the risk of prostateCancer in the patient. In one aspect of the invention the measuring ofPSA and Cln101 levels are done simultaneously. In another aspect of theinvention the measuring of PSA and Cln101 are done sequentially.

In yet another aspect of the invention, the respective levels of PSA andCln101 are based on dividing a patient population dataset intoborderline levels of PSA and elevated levels of Cln101 and a patienthaving both borderline PSA and high Cln101 levels is indicative ofheightened risk of prostate cancer. The borderline levels of PSA may bebetween about 2 ng/mL and about 10 ng/mL. The borderline levels of PSAmay also between about 4 ng/mL and about 10 ng/mL or between about 2ng/mL and about 4 ng/mL.

In addition, the invention is directed to a method of detecting ovariancancer.

The invention is also directed to a method for treating a subject withelevated risk of a prostate cancer, comprising: selecting a subject whohas borderline levels of Prostate Specific Antigen (PSA) and elevatedlevels of Cln101 and treating the subject with a therapy selected fromthe group consisting of surgery, radiation therapy, hormone therapy orchemotherapy so as to alleviate the elevated risk of prostate cancer inthe subject. The invention is also directed to a method for treating asubject with elevated risk of a Prostate Cancer, comprising: selecting asubject who has borderline levels of Prostate Specific Antigen (PSA) andelevated levels of Cln101 and treating the subject with a therapyselected from the group consisting of surgery, radiation therapy,hormone therapy or chemotherapy so as to alleviate the elevated risk ofprostate cancer in the subject.

Lastly the invention is directed to a kit for diagnosing a patient'ssusceptibility to prostate cancer comprising both a suitable assay formeasuring Cln101 levels and a suitable assay for measuring ProstateSpecific Antigen (PSA) levels wherein the levels of both PSA and Cln101are determined.

This invention is directed to an isolated Cln101 antibody that binds toCln101 on a mammalian cell in vivo. The invention is further directed toan isolated Cln101 antibody that internalizes upon binding to Cln101 ona mammalian cell in vivo. The antibody may be a monoclonal antibody.Alternatively, the antibody is an antibody fragment or a chimeric or ahumanized antibody. The monoclonal antibody may be produced by ahybridoma selected from the group of hybridomas deposited under AmericanType Culture Collection accession number PTA-5877 and PTA-5876.

The antibody may compete for binding to the same epitope as the epitopebound by the monoclonal antibody produced by a hybridoma selected fromthe group of hybridomas deposited under the American Type CultureCollection accession number PTA-5877 and PTA-5876.

The invention is also directed to conjugated antibodies. They may beconjugated to a growth inhibitory agent or a cytotoxic agent. Thecytotoxic agent may be selected from the group consisting of toxins,antibiotics, radioactive isotopes and nucleolytic enzymes and toxins.Examples of toxins include, but are not limited to, maytansin,maytansinoids, saporin, gelonin, ricin or calicheamicin.

The mammalian cell may be a cancer cell. Preferably, the anti-Cln101monoclonal antibody that inhibits the growth of Cln101-expressing cancercells in vivo.

The antibody may be produced in bacteria. Alternatively, the antibodymay be a humanized form of an anti-Cln101 antibody produced by ahybridoma selected from the group of hybridomas having ATCC accessionnumber PTA-5877 and PTA-5876.

Preferably, the cancer is selected from the group consisting of prostateand ovarian cancer. The invention is also directed to a method ofproducing the antibodies comprising culturing an appropriate cell andrecovering the antibody from the cell culture.

The invention is also directed to compositions comprising the antibodiesand a carrier. The antibody may be conjugated to a cytotoxic agent. Thecytotoxic agent may be a radioactive isotope or other chemotherapeuticagent.

The invention is also directed to a method of killing aCln101-expressing cancer cell, comprising contacting the cancer cellwith the antibodies of this invention, thereby killing the cancer cell.The cancer cell may be selected from the group consisting of prostateand ovarian cancer cell.

The prostate or ovarian cancer may be ovarian serous adenocarcinoma ormetastatic cancer.

The invention is also directed to a method of alleviating aCln101-expressing cancer in a mammal, comprising administering atherapeutically effective amount of the antibodies to the mammal.

In addition, the invention is directed to an article of manufacturecomprising a container and a composition contained therein, wherein thecomposition comprises an antibody as described herein. The article ofmanufacture may also comprise an additional component, e.g., a packageinsert indicating that the composition can be used to treat prostate orovarian cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the Anti-Cln101 MAb Epitope Map.

FIG. 2 shows Detection of Cln101 in Normal and Cancer Serum Samples(primary panel).

FIG. 3 shows Detection of Cln101 in Normal and Cancer Serum Samples(primary panel), Expanded Scale.

FIG. 4 shows Detection of Cln101 in Normal and Cancer Serum Samples(multiple panels).

FIG. 5 shows Detection of Cln101 in Normal and Cancer Serum Samples(multiple panels), Expanded Scale.

FIG. 6 shows Detection of Cln101 in Normal and Cancer Serum Samples(master panel).

FIG. 7 shows Detection of Cln101 in Normal and Cancer Serum Samples(master panel), Expanded Scale.

FIG. 8 shows Detection of Cln101 in Normal, Prostate Cancer and ProstateBenign Serum Samples.

FIG. 9 shows Detection of PSA in Normal, Prostate Cancer and ProstateBenign Serum Samples.

FIG. 10 shows Detection of Cln101 in Normal, Ovarian Cancer and OvarianBenign Serum Samples

FIG. 11 shows ROC Curves for Cln101 and PSA in Prostate Cancer vs.Normal Serum Samples.

FIG. 12 shows ROC Curves for Cln101 and PSA in Prostate Cancer andBenign Disease vs. Normal Serum Samples.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Techniques

Human “Cln101” as used herein, refers to a protein of 158 amino acidsthat is secreted from cells. The nucleotide and amino acid sequences ofCln101 have been disclosed, e.g., WO200020640-A1, DIADEXUS, Human cancerspecific protein, CC-2; WO9639541-A1 HUMAN GENOME SCIENCES, Human colonspecific protein; and J. C. Hartupee et al. Isolation andcharacterization of a cDNA encoding a novel member of the humanregenerating protein family: Reg IV. Biochim Biophys Acta. 2001 April16; 1518(3):287-93. Most of the amino acids of Cln101 are understood tobe secreted from cells. Cln101 has also been disclosed in the REFSEQdatabase as: NM_(—)032044.2 (GI: 36054181) Homo sapiens regeneratingislet-derived family, member 4 (REG4, REG IV). Cln101 (REG IV) proteincontains N-terminal signal peptide and the following domains: C-typelectin domain (InterPro Accession: IPR001304) andPancreatitis-associated domain (InterPro Accession: IPR003990). TheInterPro database is accessible at the European Bioinformatics Institute(EBI) website, ebi with the extension .ac.uk of the world wide web, thecontents of which are incorporated by reference.

Cln101 as used herein include allelic variants and conservativesubstitution mutants of the protein which have Cln101 biologicalactivity.

Recently, a series of independent publications have linked Cln101 tocolorectal and gastric cancer. See Violette, S. et al. Reg IV, a newmember of the regenerating gene family, is overexpressed in colorectalcarcinomas. Int. J. Cancer 103:185-193 (2003); Kamarainen, M. et al.RELP, a novel human REG-like protein with up-regulated expression ininflammatory and metaplastic gastrointestinal mucosa. Am. J. Pathol.163:11-20 (2003); Yonemura, Y. et al. REG gene expression is associatedwith the infiltrating growth of gastric carcinoma. Cancer 98:1394-1400(2003); Zhang, Y. et al. Overexpression of Reg IV in colorectal adenoma.Cancer Lett. 200:69-76 (2003).

Our findings that Cln101 is apparently associated to prostate andovarian cancers make this antigen an attractive target for detection andimmunotherapy of these and possibly other tumor types.

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g. bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity. The term “immunoglobulin” (Ig) is usedinterchangeably with “antibody” herein.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. Preferably, the antibody will be purified (1)to greater than 95% by weight of antibody as determined by the Lowrymethod, and most preferably more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomassie blue or, preferably, silver stain. Isolated antibody includesthe antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(VH) followed by three constant domains (CH) for each of the α, δ and γchains and four CH domains for μ and ε isotypes. Each L chain has at theN-terminus, a variable domain (VL) followed by a constant domain (CL) atits other end.

The VL is aligned with the VH and the CL is aligned with the firstconstant domain of the heavy chain (CHI). Particular amino acid residuesare believed to form an interface between the light chain and heavychain variable domains. The pairing of a VH and VL together forms asingle antigen-binding site. For the structure and properties of thedifferent classes of antibodies, see, e.g., Basic and ClinicalImmunology, 8th edition, Daniel P. Stites, Abba I. Teff and Tristram G.Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 andChapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (CH),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, γ and μ, respectively. The γ and α classesare further divided into subclasses on the basis of relatively minordifferences in C_(H) sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and define specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 1-10-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aP-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the P-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (LI), 5056 (L2) and 89-97 (L3) in the VL, and aroundabout 1-35 (HI), 50-65 (H2) and 95-102 (113) in the VH; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (e.g. residues 26-32 (LI),50-52 (L2) and 91-96 (U) in the VL, and 26-32 (HI), 53-55 (1-12) and96-101 (H3) in the VH; Chothia and Lesk J. Mol. Biol. 196:901-917(1987)).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.Old World Monkey, Ape etc), and human constant region sequences.

An “intact” antibody is one which comprises an antigen-binding site aswell as a CL and at least heavy chain constant domains, CHI, CH2 andCH3. The constant domains may be native sequence constant domains (e.g.human native sequence constant domains) or amino acid sequence variantthereof. Preferably, the intact antibody has one or more effectorfunctions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments. Papain digestion of antibodies produces twoidentical antigen-binding fragments, called “Fab” fragments, and aresidual “Fc” fragment, a designation reflecting the ability tocrystallize readily. The Fab fragment consists of an entire L chainalong with the variable region domain of the H chain (VH), and the firstconstant domain of one heavy chain (CHI). Each Fab fragment ismonovalent with respect to antigen binding, i.e., it has a singleantigen-binding site. Pepsin treatment of an antibody yields a singlelarge F(ab′)2 fragment which roughly corresponds to two disulfide linkedFab fragments having divalent antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having additional few residues at the carboxy terminus ofthe CHI domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)2antibody fragments originally were produced as pairs of 8 Fab′ fragmentswhich have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, which region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the VH and VL domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the VH and VL domains of the twoantibodies are present on different polypeptide chains. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

A “native sequence” polypeptide is one which has the same amino acidsequence as a polypeptide (e.g., antibody) derived from nature. Suchnative sequence polypeptides can be isolated from nature or can beproduced by recombinant or synthetic means. Thus, a native sequencepolypeptide can have the amino acid sequence of a naturally occurringhuman polypeptide, murine polypeptide, or polypeptide from any othermammalian species.

The term “amino acid sequence variant” refers to a polypeptide that hasamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants of Cln101 willpossess at least about 70% homology with the native sequence Cln101,preferably, at least about 80%, more preferably at least about 85%, evenmore preferably at least about 90% homology, and most preferably atleast 95%. The amino acid sequence variants can possess substitutions,deletions, and/or insertions at certain positions within the amino acidsequence of the native amino acid sequence.

The phrase “functional fragment or analog” of an antibody is a compoundhaving qualitative biological activity in common with a full-lengthantibody. For example, a functional fragment or analog of an anti-IgEantibody is one which can bind to an IgE immunoglobulin in such a mannerso as to prevent or substantially reduce the ability of such moleculefrom having the ability to bind to the high affinity receptor, FcεRI.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. Sequence similarity may be measured by any common sequence analysisalgorithm, such as GAP or BESTFIT or other variation Smith-Watermanalignment. See, T. F. Smith and M. S. Waterman, J. Mol. Biol.147:195-197 (1981) and W. R. Pearson, Genomics 11:635-650 (1991).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

As used herein, an anti-Cln101 antibody that “internalizes” is one thatis taken up by (i.e., enters) the cell upon binding to Cln101 on amammalian cell (i.e. cell surface Cln101). The internalizing antibodywill of course include antibody fragments, human or humanized antibodyand antibody conjugate. For therapeutic applications, internalization invivo is contemplated. The number of antibody molecules internalized willbe sufficient or adequate to kill a Cln101-expressing cell, especially aCln101 expressing cancer cell. Depending on the potency of the antibodyor antibody conjugate, in some instances, the uptake of a singleantibody molecule into the cell is sufficient to kill the target cell towhich the antibody binds. For example, certain toxins are highly potentin killing such that internalization of one molecule of the toxinconjugated to the antibody is sufficient to kill the tumor cell.

Whether an anti-Cln101 antibody internalizes upon binding Cln101 on amammalian cell can be determined by various assays including thosedescribed in the experimental examples below. For example, to testinternalization in vivo, the test antibody is labeled and introducedinto an animal known to have Cln101 expressed on the surface of certaincells. The antibody can be radiolabeled or labeled with fluorescent orgold particles, for instance. Animals suitable for this assay include amammal such as a NCR nude mouse that contains a human Cln101-expressingtumor transplant or xenograft, or a mouse into which cells transfectedwith human Cln101 have been introduced, or a transgenic mouse expressingthe human Cln101 transgene. Appropriate controls include animals thatdid not receive the test antibody or that received an unrelatedantibody, and animals that received an antibody to another antigen onthe cells of interest, which antibody is known to be internalized uponbinding to the antigen. The antibody can be administered to the animal,e.g., by intravenous injection. At suitable time intervals, tissuesections of the animal can be prepared using known methods or asdescribed in the experimental examples below, and analyzed by lightmicroscopy or electron microscopy, for internalization as well as thelocation of the internalized antibody in the cell. For internalizationin vitro, the cells can be incubated in tissue culture dishes in thepresence or absence of the relevant antibodies added to the culturemedia and processed for microscopic analysis at desired time points. Thepresence of an internalized, labeled antibody in the cells can bedirectly visualized by microscopy or by autoradiography if radiolabeledantibody is used. Alternatively, in a quantitative biochemical assay, apopulation of cells comprising Cln101-expressing cells are contacted invitro or in vivo with a radiolabeled test antibody and the cells (ifcontacted in vivo, cells are then isolated after a suitable amount oftime) are treated with a protease or subjected to an acid wash to removeuninternalized antibody on the cell surface. The cells are ground up andthe amount of protease resistant, radioactive counts per minute (cpm)associated with each batch of cells is measured by passing thehomogenate through a scintillation counter.

Based on the known specific activity of the radiolabeled antibody, thenumber of antibody molecules internalized per cell can be deduced fromthe scintillation counts of the ground-up cells. Cells are “contacted”with antibody in vitro preferably in solution form such as by adding thecells to the cell culture media in the culture dish or flask and mixingthe antibody well with the media to ensure uniform exposure of the cellsto the antibody. Instead of adding to the culture media, the cells canbe contacted with the test antibody in an isotonic solution such as PBSin a test tube for the desired time period. In vivo, the cells arecontacted with antibody by any suitable method of administering the testantibody such as the methods of administration described below whenadministered to a patient.

The faster the rate of internalization of the antibody upon binding tothe Cln101-expressing cell in vivo, the faster the desired killing orgrowth inhibitory effect on the target Cln101-expressing cell can beachieved, e.g., by a cytotoxic immunoconjugate. Preferably, the kineticsof internalization of the anti-Cln101 antibodies are such that theyfavor rapid killing of the Cln101-expressing target cell. Therefore, itis desirable that the anti-Cln101 antibody exhibit a rapid rate ofinternalization preferably, within 24 hours from administration of theantibody in vivo, more preferably within about 12 hours, even morepreferably within about 30 minutes to 1 hour, and most preferably,within about 30 minutes. The present invention provides antibodies thatinternalize as fast as about 15 minutes from the time of introducing theanti-Cln101 antibody in vivo. The antibody will preferably beinternalized into the cell within a few hours upon binding to Cln101 onthe cell surface, preferably within 1 hour, even more preferably within15-30 minutes.

To determine if a test antibody can compete for binding to the sameepitope as the epitope bound by the anti-Cln101 antibodies of thepresent invention including the antibodies produced by the hybridomasdeposited with the ATCC, a cross-blocking assay e.g., a competitiveELISA assay can be performed. In an exemplary competitive ELISA assay,Cln101-coated wells of a microtiter plate, or Cln101-coated sepharosebeads, are pre-incubated with or without candidate competing antibodyand then a biotin-labeled anti-Cln101 antibody of the invention isadded. The amount of labeled anti-Cln101 antibody bound to the Cln101antigen in the wells or on the beads is measured using avidin-peroxidaseconjugate and appropriate substrate.

Alternatively, the anti-Cln101 antibody can be labeled, e.g., with aradioactive or fluorescent label or some other detectable and measurablelabel. The amount of labeled anti-Cln101 antibody that binds to theantigen will have an inverse correlation to the ability of the candidatecompeting antibody (test antibody) to compete for binding to the sameepitope on the antigen, i.e., the greater the affinity of the testantibody for the same epitope, the less labeled anti-Cln101 antibodywill be bound to the antigen-coated wells. A candidate competingantibody is considered an antibody that binds substantially to the sameepitope or that competes for binding to the same epitope as ananti-Cln101 antibody of the invention if the candidate competingantibody can block binding of the anti-Cln101 antibody by at least 20%,preferably by at least 20-50%, even more preferably, by at least 50% ascompared to a control performed in parallel in the absence of thecandidate competing antibody (but may be in the presence of a knownnoncompeting antibody). It will be understood that variations of thisassay can be performed to arrive at the same quantitative value.

An antibody having a “biological characteristic” of a designatedantibody, such as any of the monoclonal antibodies Cln101.A1.1,Cln101.A3.1, Cln101.A6.1, Cln101.A7.1, Cln101.A8.1, Cln101.A9.1,Cln101.A10.1, Cln101.A11.1, Cln101.A15.1, Cln101.A16, Cln101.A17.1,Cln101.A18.1, Cln101.A23.1, Cln101.A26.1, Cln101.A28.1, Cln101.A35.1,Cln101.A37.1, Cln101.A38.1, Cln101.A41.1, Cln101.A42.1, Cln101.A46.1,Cln101.A47.1, Cln101.C3, Cln101.C6, Cln101.C12, Cln101.C17, Cln101.C18,Cln101.C25, Cln101.C36, Cln101.C43 and Cln101.C47, is one whichpossesses one or more of the biological characteristics of that antibodywhich distinguish it from other antibodies that bind to the sameantigen, Cln101.A1.1, Cln101.A3.1, Cln101.A6.1, Cln101.A7.1,Cln101.A8.1, Cln101.A9.1, Cln101.A10.1, Cln101.A11.1, Cln101.A15.1,Cln101.A16, Cln101.A17.1, Cln101.A18.1, Cln101.A23.1, Cln101.A26.1,Cln101.A28.1, Cln101.A35.1, Cln101.A37.1, Cln101.A38.1, Cln101.A41.1,Cln101.A42.1, Cln101.A46.1, Cln101.A47.1, Cln101.C3, Cln101.C6,Cln101.C12, Cln101.C17, Cln101.C18, Cln101.C25, Cln101.C36, Cln101.C43and Cln101.C47 will bind the same epitope as that bound by Cln101.A1.1,Cln101.A3.1, Cln101.A6.1, Cln101.A7.1, Cln101.A8.1, Cln101.A9.1,Cln101.A10.1, Cln101.A11.1, Cln101.A15.1, Cln101.A16, Cln101.A17.1,Cln101.A18.1, Cln101.A23.1, Cln101.A26.1, Cln101.A28.1, Cln101.A35.1,Cln101.A37.1, Cln101.A38.1, Cln101.A41.1, Cln101.A42.1, Cln101.A46.1,Cln101.A47.1, Cln101.C3, Cln101.C6, Cln101.C12, Cln101.C17, Cln101.C18,Cln101.C25, Cln101.C36, Cln101.C43 and Cln101.C47 (e.g. which competesfor binding or blocks binding of monoclonal antibody Cln101.A1.1,Cln101.A3.1, Cln101.A6.1, Cln101.A7.1, Cln101.A8.1, Cln101.A9.1,Cln101.A10.1, Cln101.A11.1, Cln101.A15.1, Cln101.A16, Cln101.A17.1,Cln101.A18.1, Cln101.A23.1, Cln101.A26.1, Cln101.A28.1, Cln101.A35.1,Cln101.A37.1, Cln101.A38.1, Cln101.A41.1, Cln101.A42.1, Cln101.A46.1,Cln101.A47.1, Cln101.C3, Cln101.C6, Cln101.C12, Cln101.C17, Cln101.C18,Cln101.C25, Cln101.C36, Cln101.C43 and Cln101.C47 to Cln101), be able totarget an Cln101-expressing tumor cell in vivo and will bind to Cln101on a mammalian cell in vivo. It is noted that the nomenclature formatfor anti-Cln101 antibodies may also be represented without the firstpunctuation point “.”. For example Cln101.A9.1 and Cln101.A46.1 may berepresented as Cln101 A9.1 and Cln101 A46.1, respectively.

Furthermore, an antibody with the biological characteristic of theCln101.A1.1, Cln101.A3.1, Cln101.A6.1, Cln101.A7.1, Cln101.A8.1,Cln101.A9.1, Cln101.A10.1, Cln101.A11.1, Cln101.A15.1, Cln101.A16,Cln101.A17.1, Cln101.A18.1, Cln101.A23.1, Cln101.A26.1, Cln101.A28.1,Cln101.A35.1, Cln101.A37.1, Cln101.A38.1, Cln101.A41.1, Cln101.A42.1,Cln101.A46.1, Cln101.A47.1, Cln101.C3, Cln101.C6, Cln101.C12,Cln101.C17, Cln101.C18, Cln101.C25, Cln101.C36, Cln101.C43 andCln101.C47 antibody will internalize upon binding to Cln101 on amammalian cell in vivo.

Likewise, an antibody with the biological characteristic of the Cln101.A1.1, Cln101.A3.1, Cln101.A6.1, Cln101.A7.1, Cln101.A8.1, Cln101.A9.1,Cln101.A10.1, Cln101.A11.1, Cln101.A15.1, Cln101.A16, Cln101.A17.1,Cln101.A18.1, Cln101.A23.1, Cln101.A26.1, Cln101.A28.1, Cln101.A35.1,Cln101.A37.1, Cln101.A38.1, Cln101.A41.1, Cln101.A42.1, Cln101.A46.1,Cln101.A47.1, Cln101.C3, Cln101.C6, Cln101.C12, Cln101.C17, Cln101.C18,Cln101.C25, Cln101.C36, Cln101.C43 and Cln101.C47 antibody will have thesame epitope binding, targeting, internalizing, tumor growth inhibitoryand cytotoxic properties of the antibody.

The term “antagonist” antibody is used in the broadest sense, andincludes an antibody that partially or fully blocks, inhibits, orneutralizes a biological activity of a native Cln101 protein disclosedherein. Methods for identifying antagonists of an Cln101 polypeptide maycomprise contacting an Cln101 polypeptide or a cell expressing Cln101 onthe cell surface, with a candidate antagonist antibody and measuring adetectable change in one or more biological activities normallyassociated with the Cln101 polypeptide.

An “antibody that inhibits the growth of tumor cells expressing Cln101”or a “growth inhibitory” antibody is one which binds to and results inmeasurable growth inhibition of cancer cells expressing oroverexpressing Cln101. Alternatively, an antibody that inhibits thegrowth of tumor cells expressing Cln101 or a growth inhibitory antibodyis one which binds to and inhibits the function of Cln101 which resultsin measurable growth inhibition of cancer cells expressing oroverexpressing Cln101. Preferred growth inhibitory anti-Cln101antibodies inhibit growth of Cln101-expressing tumor cells e.g.,prostate or ovarian cancer cells) by greater than 20%, preferably fromabout 20% to about 50%, and even more preferably, by greater than 50%(e.g. from about 50% to about 100%) as compared to the appropriatecontrol, the control typically being tumor cells not treated with theantibody being tested. Growth inhibition can be measured at an antibodyconcentration of about 0.1 to 30 pg/ml or about 0.5 nM to 200 nM in cellculture, where the growth inhibition is determined 1-10 days afterexposure of the tumor cells to the antibody. Growth inhibition of tumorcells in vivo can be determined in various ways such as is described inthe Experimental Examples section below. The antibody is growthinhibitory in vivo if administration of the anti-Cln101 antibody atabout 1 pg/kg to about 100 mg/kg body weight results in reduction intumor size or tumor cell proliferation within about 5 days to 3 monthsfrom the first administration of the antibody, preferably within about 5to 30 days.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is usually one which overexpresses Cln101. Preferably the cell is atumor cell, e.g. an prostate or ovarian cell. Various methods areavailable for evaluating the cellular events associated with apoptosis.For example, phosphatidyl serine (PS) translocation can be measured byannexin binding; DNA fragmentation can be evaluated through DNAladdering; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.Preferably, the antibody which induces apoptosis is one which results inabout 2 to 50 fold, preferably about 5 to 50 fold, and most preferablyabout 10 to 50 fold, induction of annexin binding relative to untreatedcells in an annexin binding assay.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 may be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al. PNAS (USA) 95:652-656(1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRI1B contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126.330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer, ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g. from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996) may be performed.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer ofthe urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,melanoma, multiple myeloma and B-cell lymphoma, brain, as well as headand neck cancer, and associated metastases.

A “Cln101-expressing cell” is a cell which expresses secreted endogenousor transfected Cln101. A “Cln101-expressing cancer” is a cancercomprising cells that secrete Cln101 protein from the cells. A“Cln101-expressing cancer” produces sufficient levels of Cln101 suchthat an anti-Cln101 antibody can bind thereto and have a therapeuticeffect with respect to the cancer. A cancer which “overexpresses” Cln101is one which has significantly higher levels of Cln101 compared to anoncancerous cell of the same tissue type. Such overexpression may becaused by gene amplification or by increased transcription ortranslation. Cln101 overexpression may be determined in a diagnostic orprognostic assay by evaluating increased levels of the Cln101 protein(e.g. via an immunohistochemistry assay, FACS analysis). Alternatively,or additionally, one may measure levels of Cln101-encoding nucleic acidor mRNA in the cell, e.g. via fluorescent in situ hybridization; (FISH;see WO98/45479 published October, 1998), Southern blotting, Northernblotting, or polymerase chain reaction (PCR) techniques, such as realtime quantitative PCR (RT-PCR). One may also study Cln101 overexpressionby measuring the antigen in a biological fluid such as serum, e.g.,using antibody-based assays (see also, e.g., U.S. Pat. No. 4,933,294issued Jun. 12, 1990; WO91/05264 published Apr. 18, 1991; U.S. Pat. No.5,401,638 issued Mar. 28, 1995; and Sias et al. J. Immunol. Methods 132:73-80 (1990)). Biological or bodily fluids further include blood, serum,salvia, urine, sputum, seminal fluids, and other bodily excretions suchas stool. Aside from the above assays, various in vivo assays areavailable to the skilled practitioner. For example, one may expose cellsor tissues within the body of the patient to an antibody which isoptionally labeled with a detectable label, e.g. a radioactive isotope,and binding of the antibody to cells in the patient can be evaluated,e.g. by external scanning for radioactivity or by analyzing a biopsytaken from a patient previously exposed to the antibody. ACln101-expressing cancer includes ovarian, pancreatic, lung or breastcancer.

A “mammal” for purposes of treating a cancer or alleviating the symptomsof cancer, refers to any mammal, including-humans, domestic and farmanimals, and zoo, sports, or pet animals, such as dogs, cats, cattle,horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal ishuman.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. Those in need of treatment include those already with thedisorder as well as those prone to have the disorder or those in whomthe disorder is to be prevented. A subject or mammal is successfully“treated” for an Cln101-expressing cancer if, after receiving atherapeutic amount of an anti-Cln101 antibody according to the methodsof the present invention, the patient shows observable and/or measurablereduction in or absence of one or more of the following: reduction inthe number of cancer cells or absence of the cancer cells; reduction inthe tumor size; inhibition (i.e., slow to some extent and preferablystop) of cancer cell infiltration into peripheral organs including thespread of cancer into soft tissue and bone; inhibition (i.e., slow tosome extent and preferably stop) of tumor metastasis; inhibition, tosome extent, of tumor growth; and/or relief to some extent, one or moreof the symptoms associated with the specific cancer; reduced morbidityand mortality, and improvement in quality of life issues. To the extentthe anti-Cln101 antibody may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic. Reduction of these signsor symptoms may also be felt by the patient.

The above parameters for assessing successful treatment and improvementin the disease are readily measurable by routine procedures familiar toa physician. For cancer therapy, efficacy can be measured, for example,by assessing the time to disease progression (TTP) and/or determiningthe response rate (RR).

The term “therapeutically effective amount” refers to an amount of anantibody or a drug effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the drug may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the cancer. See preceding definition of“treating”. To the extent the drug may prevent growth and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.

“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed.

Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, e.g., gelonin,ricin, saporin, and the various antitumor or anticancer agents disclosedbelow. Other cytotoxic agents are described below. A tumoricidal agentcauses destruction of tumor cells.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially anCln101-expressing cancer cell, either in vitro or in vivo. Thus, thegrowth inhibitory agent may be one which significantly reduces thepercentage of Cln101-expressing cells in S phase. Examples of growthinhibitory agents include agents that block cell cycle progression (at aplace other than S phase), such as agents that induce GI arrest andM-phase arrest. Classical M-phase blockers include the vincas(vincristine and vinblastine), taxanes, and topoisomerase II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest GI also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WBSaunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxeland docetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Label” as used herein refers to a detectable compound or compositionwhich is conjugated directly or indirectly to the antibody so as togenerate a “labeled” antibody. The label may be detectable by itself(e.g. radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

The term “epitope tagged” used herein refers to a chimeric polypeptidecomprising an anti-Cln101 antibody polypeptide fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the Ig polypeptide to whichit is fused. The tag polypeptide is also preferably fairly unique sothat the antibody does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least six aminoacid residues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

An “isolated nucleic acid molecule” is a nucleic acid molecule, e.g., anRNA, DNA, or a mixed polymer, which is substantially separated fromother genome DNA sequences as well as proteins or complexes such asribosomes and polymerases, which naturally accompany a native sequence.The term embraces a nucleic acid molecule which has been removed fromits naturally occurring environment, and includes recombinant or clonedDNA isolates and chemically synthesized analogues or analoguesbiologically synthesized by heterologous systems. A substantially purenucleic acid molecule includes isolated forms of the nucleic acidmolecule.

“Vector” includes shuttle and expression vectors and includes, e.g., aplasmid, cosmid, or phagemid. Typically, a plasmid construct will alsoinclude an origin of replication (e.g., the ColEl origin of replication)and a selectable marker (e.g., ampicillin or tetracycline resistance),for replication and selection, respectively, of the plasmids inbacteria. An “expression vector” refers to a vector that contains thenecessary control sequences or regulatory elements for expression of theantibodies including antibody fragment of the invention, in prokaryotic,e.g., bacterial, or eukaryotic cells. Suitable vectors are disclosedbelow.

The cell that produces an anti-Cln101 antibody of the invention willinclude the parent hybridoma cell e.g., the hybridomas that aredeposited with the ATCC, as well as bacterial and eukaryotic host cellsinto which nucleic acid encoding the antibodies have been introduced.Suitable host cells are disclosed below.

RNA interference refers to the process of sequence-specific posttranscriptional gene silencing in animals mediated by short interferingRNAs (siRNA) (Fire et al., 1998, Nature, 391, 806). The correspondingprocess in plants is commonly referred to as post transcriptional genesilencing or RNA silencing and is also referred to as quelling in fungi.The process of post transcriptional gene silencing is thought to be anevolutionarily conserved cellular defense mechanism used to prevent theexpression of foreign genes which is commonly shared by diverse floraand phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protectionfrom foreign gene expression may have evolved in response to theproduction of double stranded RNAs (dsRNA) derived from viral infectionor the random integration of transposon elements into a host genome viaa cellular response that specifically destroys homologous singlestranded RNA or viral genomic RNA. The presence of dsRNA in cellstriggers the RNAi response though a mechanism that has yet to be fullycharacterized. This mechanism appears to be different from theinterferon response that results from dsRNA mediated activation ofprotein kinase PKR and 2′,5′-oligoadenylate synthetase resulting innon-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from dicer activity are typically about21-23 nucleotides in length and comprise about 19 base pair duplexes.Dicer has also been implicated in the excision of 21 and 22 nucleotidesmall temporal RNAs (stRNA) from precursor RNA of conserved structurethat are implicated in translational control (Hutvagner et al., 2001,Science, 293, 834). The RNAi response also features an endonucleasecomplex containing a siRNA, commonly referred to as an RNA-inducedsilencing complex (RISC), which mediates cleavage of single stranded RNAhaving sequence complementary to the antisense strand of the siRNAduplex. Cleavage of the target RNA takes place in the middle of theregion complementary to the antisense strand of the siRNA duplex(Elbashir et al., 2001, Genes Dev., 15, 188).

Short interfering RNA mediated RNAi has been studied in a variety ofsystems. Fire et al., 1998, Nature, 391, 806, were the first to observeRNAi in C. Elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70,describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000,Nature, 404, 293, describe RNAi in Drosophila cells transfected withdsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced byintroduction of duplexes of synthetic 21-nucleotide RNAs in culturedmammalian cells including human embryonic kidney and HeLa cells. Recentwork in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J.,20, 6877) has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two nucleotide3′-overhangs. Furthermore, complete substitution of one or both siRNAstrands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAiactivity, whereas substitution of the 3′-terminal siRNA overhangnucleotides with deoxy nucleotides (2′-H) was shown to be tolerated.Single mismatch sequences in the center of the siRNA duplex were alsoshown to abolish RNAi activity. In addition, these studies also indicatethat the position of the cleavage site in the target RNA is defined bythe 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashiret al., 2001, EMBO J., 20, 6877). Other studies have indicated that a5′-phosphate on the target-complementary strand of a siRNA duplex isrequired for siRNA activity and that ATP is utilized to maintain the5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-overhanging segments of a21-mer siRNA duplex having 2 nucleotide 3′ overhangs withdeoxyribonucleotides does not have an adverse effect on RNAi activity.Replacing up to 4 nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition,Elbashir et al., supra, also report that substitution of siRNA with2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al.,International PCT Publication No. WO 00/44914, and Beach et al.,International PCT Publication No. WO 01/68836 both suggest that siRNA“may include modifications to either the phosphate-sugar back bone orthe nucleoside to include at least one of a nitrogen or sulfurheteroatom”, however neither application teaches to what extent thesemodifications are tolerated in siRNA molecules nor provide any examplesof such modified siRNA. Kreutzer and Limmer, Canadian Patent ApplicationNo. 2,359,180, also describe certain chemical modifications for use indsRNA constructs in order to counteract activation of doublestranded-RNA-dependent protein kinase PKR, specifically 2′-amino or2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer and Limmer similarly fail to show towhat extent these modifications are tolerated in siRNA molecules nor dothey provide any examples of such modified siRNA.

Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that “RNAs with two (phosphorothioate) modified bases also hadsubstantial decreases in effectiveness as RNAi triggers (data notshown); (phosphorothioate) modification of more than two residuesgreatly destabilized the RNAs in vitro and we were not able to assayinterference activities.” Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and observed that substituting deoxynucleotides forribonucleotides “produced a substantial decrease in interferenceactivity”, especially in the case of Uridine to Thymidine and/orCytidine to deoxy-Cytidine substitutions. Id. In addition, the authorstested certain base modifications, including substituting 4-thiouracil,5-bromouracil, 5-iodouracil, 3-(aminoallyl)uracil for uracil, andinosine for guanosine in sense and antisense strands of the siRNA, andfound that whereas 4-thiouracil and 5-bromouracil were all welltolerated, inosine “produced a substantial decrease in interferenceactivity” when incorporated in either strand. Incorporation of5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resultedin substantial decrease in RNAi activity as well.

Beach et al., International PCT Publication No. WO 01/68836, describesspecific methods for attenuating gene expression using endogenouslyderived dsRNA. Tuschl et al., International PCT Publication No. WO01/75164, describes a Drosophila in vitro RNAi system and the use ofspecific siRNA molecules for certain functional genomic and certaintherapeutic applications; although Tuschl, 2001, Chem. Biochem., 2,239-245, doubts that RNAi can be used to cure genetic diseases or viralinfection due “to the danger of activating interferon response”. Li etal., International PCT Publication No. WO 00/44914, describes the use ofspecific dsRNAs for use in attenuating the expression of certain targetgenes. Zernicka-Goetz et al., International PCT Publication No. WO01/36646, describes certain methods for inhibiting the expression ofparticular genes in mammalian cells using certain dsRNA molecules. Fireet al., International PCT Publication No. WO 99/32619, describesparticular methods for introducing certain dsRNA molecules into cellsfor use in inhibiting gene expression. Plaetinck et al., InternationalPCT Publication No. WO 00/01846, describes certain methods foridentifying specific genes responsible for conferring a particularphenotype in a cell using specific dsRNA molecules. Mello et al.,International PCT Publication No. WO 01/29058, describes theidentification of specific genes involved in dsRNA mediated RNAi.Deschamps Depaillette et al., International PCT Publication No. WO99/07409, describes specific compositions consisting of particular dsRNAmolecules combined with certain anti-viral agents. Driscoll et al.,International PCT Publication No. WO 01/49844, describes specific DNAconstructs for use in facilitating gene silencing in targeted organisms.Parrish et al., 2000, Molecular Cell, 6, 1977-1087, describes specificchemically modified siRNA constructs targeting the unc-22 gene of C.elegans. Tuschl et al., International PCT Publication No. WO 02/44321,describe certain synthetic siRNA constructs.

Compositions and Methods of the Invention

The invention provides anti-Cln101 antibodies. Preferably, theanti-Cln101 antibodies bind to Cln101 in vivo or in a mammalian bodilyfluid. Alternatively, the anti-Cln101 antibodies internalize uponbinding to cell surface Cln101 on a mammalian cell. The anti-Cln101antibodies may also destroy or lead to the destruction of tumor cellsbearing or secreting Cln101.

It was not apparent that Cln101 was internalization-competent. Inaddition the ability of an antibody to internalize depends on severalfactors including the affinity, avidity, and isotype of the antibody,and the epitope that it binds. We have demonstrated herein that the cellsurface Cln101 is internalization competent upon binding by theanti-Cln101 antibodies of the invention. Additionally, it wasdemonstrated that the anti-Cln101 antibodies of the present inventioncan specifically target Cln101-expressing tumor cells in vivo andinhibit or kill these cells. These in vivo tumor targeting,internalization and growth inhibitory properties of the anti-Cln101antibodies make these antibodies very suitable for therapeutic uses,e.g., in the treatment of various cancers including prostate or ovariancancer. Internalization of the anti-Cln101 antibody is preferred, e.g.,if the antibody or antibody conjugate has an intracellular site ofaction and if the cytotoxic agent conjugated to the antibody does notreadily cross the plasma membrane (e.g., the toxin calicheamicin).Internalization is not necessary if the antibodies or the agentconjugated to the antibodies do not have intracellular sites of action,e.g., if the antibody can kill the tumor cell by ADCC or some othermechanism.

The anti-Cln101 antibodies of the invention also have variousnon-therapeutic applications. The anti-Cln101 antibodies of the presentinvention can be useful for diagnosis and staging of Cln101-expressingcancers (e.g., in radioimaging). They may be used alone or incombination with other ovarian cancer markers, including, but notlimited to, CA125, HE4 and mesothelin. The anti-Cln101 antibodies of thepresent invention may also be used alone or in combination with otherprostate cancer markers, including, but not limited to Prostate SpecificAntigen (PSA). The antibodies are also useful for purification orimmunoprecipitation of Cln101 from cells or bodily fluids, for detectionand quantitation of Cln101 in vitro, e.g. in an ELISA or a Western blot,to kill and eliminate Cln101-expressing cells from a population of mixedcells as a step in the purification of other cells. The anti-Cln101antibodies of the invention can be in the different forms encompassed bythe definition of “antibody” herein. Thus, the antibodies include fulllength or intact antibody, antibody fragments, native sequence antibodyor amino acid variants, humanized, chimeric or fusion antibodies,immunoconjugates, and functional fragments thereof. In fusionantibodies, an antibody sequence is fused to a heterologous polypeptidesequence. The antibodies can be modified in the Fc region to providedesired effector functions. As discussed in more detail in the sectionsbelow, with the appropriate Fc regions, the naked antibody bound on thecell surface can induce cytotoxicity, e.g., via antibody-dependentcellular cytotoxicity (ADCC) or by recruiting complement in complementdependent cytotoxicity, or some other mechanism. Alternatively, where itis desirable to eliminate or reduce effector function, so as to minimizeside effects or therapeutic complications, certain other Fc regions maybe used.

The antibody may compete for binding, or binds substantially to, thesame epitope bound by the antibodies of the invention. Antibodies havingthe biological characteristics of the present anti-Cln101 antibodies ofthe invention are also contemplated, e.g., an anti-Cln101 antibody whichhas the biological characteristics of a monoclonal antibody produced bythe hybridomas accorded ATCC accession numbers PTA-5877 and PTA-5876,specifically including the in vivo tumor targeting, internalization andany cell proliferation inhibition or cytotoxic characteristics.Specifically provided are anti-Cln101 antibodies that bind to an epitopepresent in amino acids 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100,100-110, 110-120, 120-130, 130-140, 140-150, 150-158 of human Cln101.

Methods of producing the above antibodies are described in detail below.

The present anti-Cln101 antibodies are useful for treating aCln101-expressing cancer or alleviating one or more symptoms of thecancer in a mammal. Such a cancer includes prostate or ovarian cancer,cancer of the urinary tract, breast cancer, lung cancer, and coloncancer. Such a cancer includes more specifically, ovarian serousadenocarcinoma, breast infiltrating ductal carcinoma, prostateadenocarcinoma, renal cell carcinomas, colorectal adenocarcinomas, lungadenocarcinomas, lung squamous cell carcinomas, and pleuralmesothelioma. The breast cancer may be HER-2 negative or positive breastcancer. The cancers encompass metastatic cancers of any of thepreceding, e.g., prostate or ovarian cancer metastases. The antibody isable to bind to at least a portion of the cancer cells that expressCln101 in the mammal and preferably is one that does not induce or thatminimizes HAMA response. Preferably, the antibody is effective todestroy or kill Cln101-expressing tumor cells or inhibit the growth orproliferation of such tumor cells, in vitro or in vivo, upon binding toCln101 on the cell. Alternatively, the antibody is effective to destroyor kill Cln101-expressing tumor cells or inhibit the growth orproliferation of such tumor cells, in vitro or in vivo, upon binding tosecreted Cln101 in proximity to such tumor cells. Such an antibodyincludes a naked anti-Cln101 antibody (not conjugated to any agent).Such naked anti-Cln101 antibodies may inhibit Cln101 function oractivity upon binding. Naked anti-Cln101 antibodies having tumor growthinhibition properties in vivo include the antibodies described in theExperimental Examples below. Naked antibodies that have cytotoxic orcell growth inhibition properties can be further conjugated with acytotoxic agent to render them even more potent in tumor celldestruction. Cytotoxic properties can be conferred to an anti-Cln101antibody by, e.g., conjugating the antibody with a cytotoxic agent, toform an immunoconjugate as described below. The cytotoxic agent or agrowth inhibitory agent is preferably a small molecule. Toxins such asmaytansin, maytansinoids, saporin, gelonin, ricin or calicheamicin andanalogs or derivatives thereof, are preferable.

The invention provides a composition comprising an anti-Cln101 antibodyof the invention, and a carrier. For the purposes of treating cancer,compositions can be administered to the patient in need of suchtreatment, wherein the composition can comprise one or more anti-Cln101antibodies present as an immunoconjugate or as the naked antibody.Further, the compositions can comprise these antibodies in combinationwith other therapeutic agents such as cytotoxic or growth inhibitoryagents, including chemotherapeutic agents. The invention also providesformulations comprising an anti-Cln101 antibody of the invention, and acarrier. The formulation may be a therapeutic formulation comprising apharmaceutically acceptable carrier.

Another aspect of the invention is isolated nucleic acids encoding theanti-Cln101 antibodies. Nucleic acids encoding both the H and L chainsand especially the hypervariable region residues, chains which encodethe native sequence antibody as well as variants, modifications andhumanized versions of the antibody, are encompassed.

The invention also provides methods useful for treating aCln101-expressing cancer or alleviating one or more symptoms of thecancer in a mammal, comprising administering a therapeutically effectiveamount of an anti-Cln101 antibody to the mammal. The antibodytherapeutic compositions can be administered short term (acute) orchronic, or intermittent as directed by physician. Also provided aremethods of inhibiting the growth of, and killing a Cln101 expressingcell. Finally, the invention also provides kits and articles ofmanufacture comprising at least one antibody of this invention,preferably at least one anti-Cln101 antibody of this invention thatbinds to Cln101 in vivo or in a mammalian bodily fluid or at least oneinternalizing anti-Cln101 antibody of this invention. Kits containinganti-Cln101 antibodies find use in detecting Cln101 expression, or intherapeutic or diagnostic assays, e.g., for Cln101 cell killing assaysor for purification and/or immunoprecipitation of Cln101 from cells. Forexample, for isolation and purification of Cln101, the kit can containan anti-Cln101 antibody coupled to a solid support, e.g., a tissueculture plate or beads (e.g., sepharose beads). Kits can be providedwhich contain antibodies for detection and quantitation of Cln101 invitro, e.g. in an ELISA or a Western blot. Such antibody useful fordetection may be provided with a label such as a fluorescent orradiolabel.

Production of Anti-Cln101 Antibodies

The following describes exemplary techniques for the production of theantibodies useful in the present invention. Some of these techniques aredescribed further in Example 1. The Cln101 antigen to be used forproduction of antibodies may be, e.g., the full length polypeptide or aportion thereof, including a soluble form of Cln101 lacking the signalpeptide, a soluble form of recombinant Cln101 with a signal peptide orsynthetic peptides to selected portions of the protein.

Alternatively, cells expressing Cln101 at their cell surface (e.g. CHOor NIH-3T3 cells transformed to overexpress Cln101; ovarian, pancreatic,lung, breast or other Cln101-expressing tumor cell line), or membranesprepared from such cells can be used to generate antibodies. Thenucleotide and amino acid sequences of human and murine Cln101 areavailable as provided above. Cln101 can be produced recombinantly in andisolated from, prokaryotic cells, e.g., bacterial cells, or eukaryoticcells using standard recombinant DNA methodology. Cln101 can beexpressed as a tagged (e.g., epitope tag) or other fusion protein tofacilitate its isolation as well as its identification in variousassays.

Antibodies or binding proteins that bind to various tags and fusionsequences are available as elaborated below. Other forms of Cln101useful for generating antibodies will be apparent to those skilled inthe art.

Tags

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (Field et al., Mol. Cell. Biol., 8:2159-2165(1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990)). The FLAG-peptide (Hopp et al., BioTechnology, 6:1204-1210(1988)) is recognized by an anti-FLAG M2 monoclonal antibody (EastmanKodak Co., New Haven, Conn.). Purification of a protein containing theFLAG peptide can be performed by immunoaffinity chromatography using anaffinity matrix comprising the anti-FLAG M2 monoclonal antibodycovalently attached to agarose (Eastman Kodak Co., New Haven, Conn.).Other tag polypeptides include the KT3 epitope peptide [Martin et al.,Science, 255:192-194 (1992)]; an α-tubulin epitope peptide (Skinner etal., J. Biol. Chenz., 266:15163-15166 (1991)); and the T7 gene proteinpeptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)).

Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals, preferablynon-human animals, by multiple subcutaneous (sc) or intraperitoneal (ip)injections of the relevant antigen and an adjuvant. It may be useful toconjugate the relevant antigen (especially when synthetic peptides areused) to a protein that is immunogenic in the species to be immunized.For example, the antigen can be conjugated to keyhole limpet hemocyanin(KLH), serum, bovine thyroglobulin, or soybean trypsin inhibitor, usinga bifunctional or derivatizing agent, e.g., maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraidehyde, succinicanhydride, SOCl₂, or R¹N═C═NR, where R and R¹ are different alkylgroups. Conjugates also can be made in recombinant cell culture asprotein fusions.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 5-100 pg of the protein or conjugate(for rabbits or mice, respectively) with 3 volumes of Freund's completeadjuvant and injecting the solution intradermally at multiple sites. Onemonth later, the animals are boosted with ⅕ to 1/10 the original amountof peptide or conjugate in Freund's complete adjuvant by subcutaneousinjection at multiple sites. Seven to 14 days later, the animals arebled and the serum is assayed for antibody titer. Animals are boosteduntil the titer plateaus. Also, aggregating agents such as alum aresuitably used to enhance the immune response.

Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567). In the hybridomamethod, a mouse or other appropriate host animal, such as a hamster, isimmunized as described above to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Alternatively, lymphocytes may beimmunized in vitro. After immunization, lymphocytes are isolated andthen fused with a “fusion partner”, e.g., a myeloma cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell (Goding, Monoclonal Antibodies. Principles and Practice, pp 103(Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, fusion partner, e.g,the parental myeloma cells. For example, if the parental myeloma cellslack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRTor HPRT), the selective culture medium for the hybridomas typically willinclude hypoxanthine, aminopterin, and thymidine (HAT medium), whichsubstances prevent the growth of HGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as those derived from MOPC-21 and MPC-IImouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cellsavailable from the American Type Culture Collection, Rockville, Md. USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980). Once hybridoma cells that produce antibodiesof the desired specificity, affinity, and/or activity are identified,the clones may be subcloned by limiting dilution procedures and grown bystandard methods (Goding, Monoclonal Antibodies: Principles andPractice, pp 103 (Academic Press, 1986)). Suitable culture media forthis purpose include, for example, D-MEM or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal e.g, by i.p. injection of the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transformed or transfected intoprokaryotic or eukaryotic host cells such as, e.g., E coli cells, simianCOS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells, that donot otherwise produce antibody protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of DNA encoding the antibody includeSkerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) andPhickthun, Immunol. Revs., 130:151-188 (1992).

Further, the monoclonal antibodies or antibody fragments can be isolatedfrom antibody phage libraries generated using the techniques describedin McCafferty et al., Nature, 348:552-554 (1990). Clackson et al.,Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597(1991) describe the isolation of murine and human antibodies,respectively, using phage libraries. Subsequent publications describethe production of high affinity (nM range) human antibodies by chainshuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nuc. Acids.Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (CH and CL) sequences for thehomologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, etal., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenonimmunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

Humanized Antibodies

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source which is nonhuman. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting hypervariable region sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity and HAMA response (human anti-mouse antibody) when theantibody is intended for human therapeutic use. According to theso-called “best-fit” method, the sequence of the variable domain of arodent antibody is screened against the entire library of known humanvariable domain sequences. The human V domain sequence which is closestto that of the rodent is identified and the human framework region (FR)within it accepted for the humanized antibody (Sims et al., J. Immunol.,151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Anothermethod uses a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh binding affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art.

Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the hypervariable region residues are directly and most substantiallyinvolved in influencing antigen binding.

Various forms of a humanized anti-Cln101 antibody are contemplated. Forexample, the humanized antibody may be an antibody fragment, such as aFab, which is optionally conjugated with one or more cytotoxic agent(s)in order to generate an immunoconjugate. Alternatively, the humanizedantibody may be an intact antibody, such as an intact IgG1 antibody.

Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669 (all of GenPharm); 5,545,807; and Alternatively, phage displaytechnology (McCafferty et al., Nature 348:552-553 (1990)) can be used toproduce human antibodies and antibody fragments in vitro, fromimmunoglobulin variable (V) domain gene repertoires from unimmunizeddonors. According to this technique, antibody V domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, such as Ml3 or fd, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. Thus, the phage mimics some of the properties of the B-cell.Phage display can be performed in a variety of formats, reviewed in,e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion inStructural Biology 3:564-571 (1993). Several sources of V-gene segmentscan be used for phage display. Clackson et al., Nature, 352:624-628(1991) isolated a diverse array of anti-oxazolone antibodies from asmall random combinatorial library of V genes derived from the spleensof immunized mice. A repertoire of V genes from unimmunized human donorscan be constructed and antibodies to a diverse array of antigens(including self-antigens) can be isolated essentially following thetechniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991),or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos.5,565,332 and 5,573,905. As discussed above, human antibodies may alsobe generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610and 5,229,275).

Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improved access tosolid tumors. Various techniques have been developed for the productionof antibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,Journal of Biochemical and Biophysical Methods 24:107-117 (1992); andBrennan et al., Science, 229:81 (1985)). However, these fragments cannow be produced directly by recombinant host cells. Fab, Fv and ScFvantibody fragments can all be expressed in and secreted from E coli,thus allowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab)2 fragments(Carter et al., Bio/Technology 10: 163-167 (1992)). According to anotherapproach, F(ab)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. The antibody of choice may also be a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat.No. 5,587,458. Fv and sFv are the only species with intact combiningsites that are devoid of constant regions; thus, they are suitable forreduced nonspecific binding during in vivo use. sFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the Cln101 protein. Other suchantibodies may combine an Cln101 binding site with a binding site foranother protein. Alternatively, an anti-Cln101.Arm may be combined withan arm which binds to a triggering molecule on a leukocyte such as aTcell receptor molecule (e.g. C133), or Fc receptors for IgG (FcγR),such as FcγRI (CD64), FcγRlI (CD32) and FcγRIII (CD16), so as to focusand localize cellular defense mechanisms to the Cln101-expressing cell.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express Cln101. These antibodies possess an Cln101-bindingarm and an arm which binds the cytotoxic agent (e.g. saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab)₂ bispecificantibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIIIantibody and U.S. Pat. No. 5,837,234 discloses a bispecificanti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcα antibody isshown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecificanti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ, 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to havethe first heavy-chain constant region (CHI) containing the sitenecessary for light chain bonding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant affect on the yield of thedesired chain combination.

Preferably, the bispecific antibodies in this approach are composed of ahybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. It was foundthat this asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. Thisapproach is disclosed in WO 94/04690. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-inking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agent,sodium arsenite, to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)2molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers.

The “diabody” technology described by Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternativemechanism for making bispecific antibody fragments. The fragmentscomprise a VH connected to a VL by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may comprise VD1(X1n-VD2-(X2)n-Fc,wherein VDI is a first variable domain, VD2 is a second variable domain,Fc is one polypeptide chain of an Fc region, X1 and X2 represent anamino acid or polypeptide, and n is 0 or 1. For instance, thepolypeptide chain(s) may comprise: VH-CHI-flexible linker-VH-CHI-Fcregion chain; or VH-CHI-VH-CHI-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain.

Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the anti-Cln101 antibodiesdescribed herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of the anti-Cln101 antibody areprepared by introducing appropriate nucleotide changes into theanti-Cln101 antibody nucleic acid, or by peptide synthesis.

Such modifications include, for example, deletions from, and/orinsertions into, and/or substitutions of, residues within the amino acidsequences of the anti-Cln101 antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe anti-Cln101 antibody, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of theanti-Cln101 antibody that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis” as described by Cunningham andWells in Science, 244:1081-1085 (1989). Here, a residue or group oftarget residues within the anti-Cln101 antibody are identified (e.g.,charged residues such as arg, asp, his, lys, and glu) and replaced by aneutral or negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with Cln101antigen.

Those amino acid locations demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other variantsat, or for, the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, toanalyze the performance of a mutation at a given site, ala scanning orrandom mutagenesis is conducted at a target codon or region and theexpressed anti-Cln101 antibody variants are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean anti-Cln101 antibody with an N-terminal methionyl residue or theantibody fused to a cytotoxic polypeptide. Other insertional variants ofthe anti-Cln101 antibody molecule include the fusion to the N- orC-terminus of the anti-Cln101 antibody to an enzyme (e.g. for ADEPT) ora fusion to a polypeptide which increases the serum half-life of theantibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the anti-Cln101antibody molecule replaced by a different residue. The sites of greatestinterest for substitutional mutagenesis include the hypervariableregions, but FR alterations are also contemplated. Conservativesubstitutions are shown in Table I under the heading of “preferredsubstitutions”. If such substitutions result in a change in biologicalactivity, then more substantial changes, denominated “exemplarysubstitutions” in Table 1, or as further described below in reference toamino acid classes, may be introduced and the products screened for adesired characteristic. TABLE I Amino Acid Substitutions OriginalExemplary Substitutions Preferred Substitutions Ala (A) val; leu; ileVal Arg (R) lys; gln; asn lys Asn (N) gln; his; asp, lys; arg gln Asp(D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp;gln asp Gly (G) ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val;met; ala; phe; leu Leu (L) norleucine; ile; val; met; ala; ile Lys (K)arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala;tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phetyr Tyr (Y) trp; phe; thr; ser Phe Val (V) ile; leu; met; phe; ala; leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutralhydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin,his, lys, arg; (5) residues that influence chain orientation: gly, pro;and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Any cysteine residue not involved inmaintaining the proper conformation of the anti-Cln101 antibody also maybe substituted, generally with serine, to improve the oxidativestability of the molecule and prevent aberrant crosslinking. Conversely,cysteine bond(s) may be added to the antibody to improve its stability(particularly where the antibody is an antibody fragment such as an Fvfragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g. 6-7 sites) are mutated togenerate all possible amino acid substitutions at each site. Theantibody variants thus generated are displayed in a monovalent fashionfrom filamentous phage particles as fusions to the gene III product ofMl3 packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and human Cln101. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to theantibody is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-Cln101 antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared nucleic acid molecule encoding a variant or a non-variantversion of the anti-Cln101 antibody.

It may be desirable to modify the antibody of the invention with respectto effector function, e.g. so as to enhance antigen-dependentcell-mediated cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al. Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of the antibody.

Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above. One mayfurther select antibodies with certain biological characteristics, asdesired.

The growth inhibitory effects of an anti-Cln101 antibody of theinvention may be assessed by methods known in the art, e.g., using cellswhich express Cln101 either endogenously or following transfection withthe Cln101 gene. For example, the tumor cell lines andCln101-transfected cells provided in Example 1 below may be treated withan anti-Cln101 monoclonal antibody of the invention at variousconcentrations for a few days (e.g., 2-7) days and stained with crystalviolet or MTT or analyzed by some other colorimetric assay. Anothermethod of measuring proliferation would be by comparing ³H-thymidineuptake by the cells treated in the presence or absence an anti-Cln101antibody of the invention. After antibody treatment, the cells areharvested and the amount of radioactivity incorporated into the DNAquantitated in a scintillation counter. Appropriated positive controlsinclude treatment of a selected cell line with a growth inhibitoryantibody known to inhibit growth of that cell line. Growth inhibition oftumor cells in vivo can be determined in various ways such as isdescribed in the Experimental Examples section below. Preferably, thetumor cell is one that over-expresses Cln101. Preferably, theanti-Cln101 antibody will inhibit cell proliferation of aCln101-expressing tumor cell in vitro or in vivo by about 25-100%compared to the untreated tumor cell, more preferably, by about 30-100%,and even more preferably by about 50-100% or 70-100%, at an antibodyconcentration of about 0.5 to 30 μg/ml. Growth inhibition can bemeasured at an antibody concentration of about 0.5 to 30 μg/ml or about0.5 nM to 200 nM in cell culture, where the growth inhibition isdetermined 1-10 days after exposure of the tumor cells to the antibody.The antibody is growth inhibitory in vivo if administration of theanti-Cln101 antibody at about 1 μg/kg to about 100 mg/kg body weightresults in reduction in tumor size or tumor cell proliferation withinabout 5 days to 3 months from the first administration of the antibody,preferably within about 5 to 30 days.

To select for antibodies which induce cell death, loss of membraneintegrity as indicated by, e.g., propidium iodide (P), trypan blue or7AAD uptake may be assessed relative to a control. A PI uptake assay canbe performed in the absence of complement and immune effector cells.Cln101-expressing tumor cells are incubated with medium alone or mediumcontaining of the appropriate monoclonal antibody at e.g., about 10μg/ml. The cells are incubated for a 3 day time period. Following eachtreatment, cells are washed and aliquoted into 35 mm strainer-capped12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal ofcell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzedusing a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software(Becton Dickinson). Those antibodies which induce statisticallysignificant levels of cell death as determined by PI uptake may beselected as cell death-inducing antibodies.

To screen for antibodies which bind to an epitope on Cln101 bound by anantibody of interest, e.g., the Cln101 antibodies of this invention, aroutine cross-blocking assay such as that describe in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed. This assay can be used to determine if atest antibody binds the same site or epitope as an anti-Cln101 antibodyof the invention. Alternatively, or additionally, epitope mapping can beperformed by methods known in the art. For example, the antibodysequence can be mutagenized such as by alanine scanning, to identifycontact residues. The mutant antibody is initially tested for bindingwith polyclonal antibody to ensure proper folding. In a differentmethod, peptides corresponding to different regions of Cln101 can beused in competition assays with the test antibodies or with a testantibody and an antibody with a characterized or known epitope.

For example, a method to screen for antibodies that bind to an epitopewhich is bound by an antibody this invention may comprise combining anCln101-containing sample with a test antibody and an antibody of thisinvention to form a mixture, the level of Cln101 antibody bound toCln101 in the mixture is then determined and compared to the level ofCln101 antibody bound in the mixture to a control mixture, wherein thelevel of Cln101 antibody binding to Cln101 in the mixture as compared tothe control is indicative of the test antibody's binding to an epitopethat is bound by the anti-Cln101 antibody of this invention. The levelof Cln101 antibody bound to Cln101 is determined by ELISA. The controlmay be a positive or negative control or both. For example, the controlmay be a mixture of Cln101, Cln101 antibody of this invention and anantibody known to bind the epitope bound by the Cln101 antibody of thisinvention. The anti-Cln101 antibody labeled with a label such as thosedisclosed herein. The Cln101 may be bound to a solid support, e.g., atissue culture plate or to beads, e.g., sepharose beads.

Immunoconjugates

The invention also pertains to therapy with immunoconjugates comprisingan antibody conjugated to an anti-cancer agent such as a cytotoxic agentor a growth inhibitory agent.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Conjugates of an antibodyand one or more small molecule toxins, such as a calicheamicin,maytansinoids, a trichothene, and CC1065, and the derivatives of thesetoxins that have toxin activity, are also contemplated herein.

Maytansine and Maytansinoids

Preferably, an anti-Cln101 antibody (full length or fragments) of theinvention is conjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the cast Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, the disclosures of which are hereby expressly incorporated byreference.

Maytansinoid-Antibody Conjugates

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens.

Immunoconjugates containing maytansinoids and their therapeutic use aredisclosed, for example, in U.S. Pat. Nos. 5,208,020, 5,416,064 andEuropean Patent EP 0 425 235 B1, the disclosures of which are herebyexpressly incorporated by reference. Liu et al., Proc. Natl. Acad. Sci.USA 93:8618-8623 (1996) described immunoconjugates comprising amaytansinoid designated DMI linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al. CancerResearch 52:127-131 (1992) describe immunoconjugates in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×10 5 HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansonid drug, which couldbe increased by increasing the number of maytansinoid molecules perantibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Anti-Cln101 Antibody-Maytansinoid Conjugates (Immunoconjugates)

Anti-Cln101 antibody-maytansinoid conjugates are prepared by chemicallylinking an anti-Cln101 antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. An average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody, although even one molecule oftoxin/antibody would be expected to enhance cytotoxicity over the use ofnaked antibody. Maytansinoids are well known in the art and can besynthesized by known techniques or isolated from natural sources.Suitable maytansinoids are disclosed, for example, in U.S. Pat. No.5,208,020 and in the other patents and nonpatent publications referredto hereinabove. Preferred maytansinoids are maytansinol and maytansinolanalogues modified in the aromatic ring or at other positions of themaytansinol molecule, such as various maytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B 1, andChari et al. Cancer Research 52: 127-131 (1992). The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred. Conjugates of the antibody and maytansinoid maybe made using a variety of bifunctional protein coupling agents such asN-succinimidyl(2-pyridyidithio)propionate (SPDP),succinimidyl-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as his(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl (2-pyridyldithio)propionate (SPDP) (Carlsson etal., Biochem. J. 173:723-737 [1978]) and N-succinimidyl(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. Preferably, the linkage is formedat the C-3 position of maytansinol or a maytansinol analogue.

Calicheamicin

Another immunoconjugate of interest comprises an anti-Cln101 antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ₁^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ₁ ^(I), (Hinman etal. Cancer Research 53: 3336 (1993), Lode et al. Cancer Research 5 8:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the anti-Cln101antibodies of the invention include BCNU, streptozoicin, vincristine and5-fluorouracil, the family of agents known collectively LL-E33288complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well asesperamicins (U.S. Pat. No. 5,877,296). Enzymatically active toxins andfragments thereof which can be used include diphtheria A chain, 1 5nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginiosa), ricin A chain, abrin A chain, modeccin Achain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordicacharantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor,gelonin, mitogellin, restrictocin, phenomycin, enomycin and thetricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993.The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g. aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated anti-Cln101 antibodies. Examplesinclude At²¹¹, I¹³¹, I¹²⁵, In¹¹¹, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³²,and radioactive isotopes of Lu. When the conjugate is used fordiagnosis, it may comprise a radioactive atom for scintigraphic studies,for example Tc⁹⁹M or I¹²³, or a spin label for nuclear magneticresonance (NMR) imaging (also known as magnetic resonance imaging, mri),such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc^(99M), I¹²³, In¹¹¹, Re¹⁸⁶, Re¹⁸⁸, can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl(2-pyridyldithio)propionate (SPDP),succinimidyl(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon labeled 1-isothiocyanatobenzyl methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al. Cancer Research 52: 127-131(1992); U.S. Pat. No. 5,208,020) may be used.

Alternatively, a fusion protein comprising the anti-Cln101 antibody andcytotoxic agent may be made, e.g. by recombinant techniques or peptidesynthesis. The length of DNA may comprise respective regions encodingthe two portions of the conjugate either adjacent one another orseparated by a region encoding a linker peptide which does not destroythe desired properties of the conjugate.

In addition, the antibody may be conjugated to a “receptor” (suchstreptavidin) for utilization in tumor pre-targeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide).

Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form. Enzymes that are useful in themethod of this invention include, but are not limited to, alkalinephosphatase useful for converting phosphate-containing prodrugs intofree drugs; arylsulfatase useful for converting sulfate-containingprodrugs into free drugs; cytosine deaminase useful for convertingnon-toxic fluorocytosine into the anti-cancer drug, 5-fluorouracil;proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidases and cathepsins (such as cathepsins B and L), that areuseful for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, useful for converting prodrugs that containD-amino acid substituents; carbohydrate-cleaving enzymes such asO-galactosidase and neuraminidase useful for converting glycosylatedprodrugs into free drugs; P-lactamase useful for converting drugsderivatized with P-lactams into free drugs; and penicillin amidases,such as penicillin V amidase or penicillin G amidase, useful forconverting drugs derivatized at their amine nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population. The enzymes of this invention can becovalently bound to the anti-Cln101 antibodies by techniques well knownin the art such as the use of the heterobifunctional crosslinkingreagents discussed above.

Alternatively, fusion proteins comprising at least the antigen bindingregion of an antibody of the invention linked to at least a functionallyactive portion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 (1984).

Other Antibody Modifications

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol. The antibody also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980).

The anti-Cln101 antibodies disclosed herein may also be formulated asimmunoliposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

Vectors, Host Cells, and Recombinant Methods

The invention also provides isolated nucleic acid molecule encoding thehumanized anti-Cln101 antibody, vectors and host cells comprising thenucleic acid, and recombinant techniques for the production of theantibody. For recombinant production of the antibody, the nucleic acidmolecule encoding it is isolated and inserted into a replicable vectorfor further cloning (amplification of the DNA) or inserted into a vectorin operable linkage with a promoter for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to nucleic acid molecules encoding the heavy andlight chains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

Signal Sequence Component

The anti-Cln101 antibody of this invention may be produced recombinantlynot only directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native anti-Cln101 antibody signal sequence,the signal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeastsecretion the native signal sequence may be substituted by, e.g., theyeast invertase leader, oc factor leader (including Saccharomyces andKluyveromyces cc-factor leaders), or acid phosphatase leader, the Calbicans glucoamylase leader, or the signal described in WO 90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable. The DNA for such precursor region is ligated in reading frameto DNA encoding the anti-Cln101 antibody.

Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theanti-Cln101 antibody nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -11, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc. For example, cellstransformed with the DHFR selection gene are first identified byculturing all of the transformants in a culture medium that containsmethotrexate (Mtx), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinese hamster ovary(CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding anti-Cln101 antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trpl gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrpl gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4 Jones, Genetics, 85:12 (1977). The presence of the trpl lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 pm circular plasmid pKDI canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to theanti-Cln101 antibody nucleic acid. Promoters suitable for use withprokaryotic hosts include the phoA promoter, P-lactamase and lactosepromoter systems, alkaline phosphatase promoter, a tryptophan (trp)promoter system, and hybrid promoters such as the tac promoter. However,other known bacterial promoters are suitable. Promoters for use inbacterial systems also will contain a Shine-Dalgarno (S.D.) sequenceoperably linked to the DNA encoding the anti-Cln101 antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors. Examples of suitable promoter sequences for use withyeast hosts include the promoters for 3-phosphoglycerate kinase or otherglycolytic enzymes, such as enolase, glyceraldehyde phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde phosphate dehydrogenase, andenzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Anti-Cln101 antibody transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIll E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human P-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

Enhancer Element Component

Transcription of a DNA encoding the anti-Cln101 antibody of thisinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theanti-Cln101 antibody-encoding sequence, but is preferably located at asite 5′ from the promoter.

Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′ untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding anti-Cln101 antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO 94/11026 and the expression vectordisclosed therein.

Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W31 10 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fragments, and antibody fusion proteinscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in tumor cell destruction.Full length antibodies have greater half life in circulation. Productionin E. coli is faster and more cost efficient. For expression of antibodyfragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), andU.S. Pat. No. 5,840,523 (Simmons et al.) which describes translationinitiation region (TIR) and signal sequences for optimizing expressionand secretion, these patents incorporated herein by reference. Afterexpression, the antibody is isolated from the E. coli cell paste in asoluble fraction and can be purified through, e.g., a protein A or Gcolumn depending on the isotype. Final purification can be carried outsimilar to the process for purifying antibody expressed e.g, in CHOcells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-Cln101antibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated anti-Cln101antibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,Arabidopsis and tobacco can also be utilized as hosts. Cloning andexpression vectors useful in the production of proteins in plant cellculture are known to those of skill in the art. See e.g. Hiatt et al.,Nature (1989) 342: 76-78, Owen et al. (1992) Bio/Technology 10: 790-794,Artsaenko et al. (1995) The Plant J 8: 745-750, and Fecker et al. (1996)Plant Mol Biol 32: 979-986.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, 1413 8065); mouse mammary tumor (MMT060562, ATCC CCL5 1); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for anti-Cln101 antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

Culturing Host Cells

The host cells used to produce the anti-Cln101 antibody of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's FIO (Sigma), Minimal Essential Medium (MEM)(Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM)(Sigma)are suitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

Purification of Anti-Cln101 Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10: 163-167 (1992) describe a procedure forisolating antibodies which are secreted to the periplasmic space of Ecoli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30min. Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SIDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Pharmaceutical Formulations

Pharmaceutical formulations of the antibodies used in accordance withthe present invention are prepared for storage by mixing an antibodyhaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as acetate, Tris,phosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol, and mcresol); low molecular weight(less than about 10 residues) polypeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyllolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; tonicifiers such as trehaloseand sodium chloride; sugars such as sucrose, mannitol, trehalose orsorbitol; surfactant such as polysorbate; salt-forming counter-ions suchas sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theantibody preferably comprises the antibody at a concentration of between5-200 mg/ml, preferably between 10-100 mg/ml.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, in addition to the anti-Cln101 antibody which internalizes,it may be desirable to include in the one formulation, an additionalantibody, e.g. a second anti-Cln101 antibody which binds a differentepitope on Cln101, or an antibody to some other target such as a growthfactor that affects the growth of the particular cancer. Alternatively,or additionally, the composition may further comprise a chemotherapeuticagent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonalagent, and/or cardioprotectant. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methylmethacylate) microcapsules, respectively,in colloidal drug delivery systems (for example, liposomes, albuminmicrospheres, microemulsions, nano-particles and nanocapsules) or inmacroemulsions. Such techniques are disclosed in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−) hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Methods and Treatment Using Anti-Cln101 Antibodies

According to the present invention, the anti-Cln101 antibody that bindsto Cln101 in vivo or internalizes upon binding Cln101 on a cell surfaceis used to treat a subject in need thereof having a cancer characterizedby Cln101-expressing cancer cells, in particular, ovarian, pancreatic,lung or breast cancer, such as ovarian serous adenocarcinoma or breastinfiltrating ductal carcinoma cancer, and associated metastases.

The cancer will generally comprise Cln101-expressing cells, such thatthe anti-Cln101 antibody is able to bind thereto. While the cancer maybe characterized by overexpression of the Cln101 molecule, the presentapplication further provides a method for treating cancer which is notconsidered to be a Cln101-overexpressing cancer.

This invention also relates to methods for detecting cells whichoverexpress Cln101 and to diagnostic kits useful in detecting cellsexpressing Cln101 or in detecting Cln101 in serum from a patient orother bodily fluids. The methods may comprise combining acell-containing test sample with an antibody of this invention, assayingthe test sample for antibody binding to cells in the test sample andcomparing the level of antibody binding in the test sample to the levelof antibody binding in a control sample of cells. A suitable control is,e.g., a sample of normal cells of the same type as the test sample or acell sample known to be free of Cln101 overexpressing cells. A level ofCln101 binding higher than that of such a control sample would beindicative of the test sample containing cells that overexpress Cln101.Alternatively the control may be a sample of cells known to containcells that overexpress Cln101. In such a case, a level of Cln101antibody binding in the test sample that is similar to, or in excess of,that of the control sample would be indicative of the test samplecontaining cells that overexpress Cln101.

Cln101 overexpression may be detected with a various diagnostic assays.For example, over expression of Cln101 may be assayed byimmunohistochemistry (IHC). Parrafin embedded tissue sections from atumor biopsy may be subjected to the IHC assay and accorded an Cln101protein staining intensity criteria as follows.

Score 0 no staining is observed or membrane staining is observed in lessthan 10% of tumor cells.

Score 1+ a faint/barely perceptible membrane staining is detected inmore than 10% of the tumor cells. The cells are only stained in part oftheir membrane.

Score 2+ a weak to moderate complete membrane staining is observed inmore than 10% of the tumor cells.

Score 3+ a moderate to strong complete membrane staining is observed inmore than 10% of the tumor cells.

Those tumors with 0 or 1+ scores for Cln101 expression may becharacterized as not overexpressing Cln101, whereas those tumors with 2+or 3+ scores may be characterized as overexpressing Cln101.

Alternatively, or additionally, FISH assays such as the INFORM™ (sold byVentana, Arizona) or PATHVISION™ (VySiS, Illinois) may be carried out onformalin-fixed, paraffin-embedded tumor tissue to determine the extent(if any) of Cln101 overexpression in the tumor. Cln101 overexpression oramplification may be evaluated using an in vivo diagnostic assay, e.g.by administering a molecule (such as an antibody of this invention)which binds Cln101 and which is labeled with a detectable label (e.g. aradioactive isotope or a fluorescent label) and externally scanning thepatient for localization of the label.

A sample suspected of containing cells expressing or overexpressingCln101 is combined with the antibodies of this invention underconditions suitable for the specific binding of the antibodies toCln101. Binding and/or internalizing the Cln101 antibodies of thisinvention is indicative of the cells expressing Cln101. The level ofbinding may be determined and compared to a suitable control, wherein anelevated level of bound Cln101 as compared to the control is indicativeof Cln101 overexpression. The sample suspected of containing cellsoverexpressing Cln101 may be a cancer cell sample, particularly a sampleof an ovarian cancer, e.g. ovarian serous adenocarcinoma, or a breastcancer, e.g., a breast infiltrating ductal carcinoma. A serum samplefrom a subject may also be assayed for levels of Cln101 by combining aserum sample from a subject with an Cln101 antibody of this invention,determining the level of Cln101 bound to the antibody and comparing thelevel to a control, wherein an elevated level of Cln101 in the serum ofthe patient as compared to a control is indicative of overexpression ofCln101 by cells in the patient. The subject may have a cancer such ase.g., an ovarian cancer, e.g. ovarian serous adenocarcinoma, or a breastcancer, e.g., a breast infiltrating ductal carcinoma.

Currently, depending on the stage of the cancer, ovarian, pancreatic,lung or breast cancer treatment involves one or a combination of thefollowing therapies: surgery to remove the cancerous tissue, radiationtherapy, androgen deprivation (e.g., hormonal therapy), andchemotherapy. Anti-Cln101 antibody therapy may be especially desirablein elderly patients who do not tolerate the toxicity and side effects ofchemotherapy well, in metastatic disease where radiation therapy haslimited usefulness, and for the management of prostatic carcinoma thatis resistant to androgen deprivation treatment. The tumor targeting andinternalizing anti-Cln101 antibodies of the invention are useful toalleviate Cln101-expressing cancers, e.g., ovarian, pancreatic, lung orbreast cancers upon initial diagnosis of the disease or during relapse.For therapeutic applications, the anti-Cln101 antibody can be usedalone, or in combination therapy with, e.g., hormones, antiangiogens, orradiolabelled compounds, or with surgery, cryotherapy, and/orradiotherapy, notably for ovarian, pancreatic, lung or breast cancers,also particularly where shed cells cannot be reached. Anti-Cln101antibody treatment can be administered in conjunction with other formsof conventional therapy, either consecutively with, pre- orpost-conventional therapy, Chemotherapeutic drugs such as Taxotere®(docetaxel), Taxol® (paclitaxel), estramustine and mitoxantrone are usedin treating metastatic and hormone refractory ovarian, pancreatic, lungor breast cancer, in particular, in good risk patients. In the presentmethod of the invention for treating or alleviating cancer, inparticular, androgen independent and/or metastatic ovarian, pancreatic,lung or breast cancer, the cancer patient can be administeredanti-Cln101 antibody in conjunction with treatment with the one or moreof the preceding chemotherapeutic agents. In particular, combinationtherapy with paclitaxel and modified derivatives (see, e.g., EP0600517)is contemplated. The anti-Cln101 antibody will be administered with atherapeutically effective dose of the chemotherapeutic agent. Theanti-Cln101 antibody may also be administered in conjunction withchemotherapy to enhance the activity and efficacy of thechemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference(PDR) discloses dosages of these agents that have been used in treatmentof various cancers. The dosing regimen and dosages of theseaforementioned chemotherapeutic drugs that are therapeutically effectivewill depend on the particular cancer being treated, the extent of thedisease and other factors familiar to the physician of skill in the artand can be determined by the physician.

Particularly, an immunoconjugate comprising the anti-Cln101 antibodyconjugated with a cytotoxic agent may be administered to the patient.Preferably, the immunoconjugate bound to the Cln101 protein isinternalized by the cell, resulting in increased therapeutic efficacy ofthe immunoconjugate in killing the cancer cell to which it binds.Preferably, the cytotoxic agent targets or interferes with the nucleicacid in the cancer cell. Examples of such cytotoxic agents are describedabove and include maytansin, maytansinoids, saporin, gelonin, ricin,calicheamicin, ribonucleases and DNA endonucleases.

The anti-Cln101 antibodies or immunoconjugates are administered to ahuman patient, in accord with known methods, such as intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time, by intramuscular, intraperitoneal, intracerebrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. The antibodies or immunoconjugates may beinjected directly into the tumor mass. Intravenous or subcutaneousadministration of the antibody is preferred. Other therapeutic regimensmay be combined with the administration of the anti-Cln101 antibody.

The combined administration includes co-administration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities. Preferably such combined therapy results in asynergistic therapeutic effect.

It may also be desirable to combine administration of the anti-Cln101antibody or antibodies, with administration of an antibody directedagainst another tumor antigen associated with the particular cancer. Assuch, this invention is also directed to an antibody “cocktail”comprising one or more antibodies of this invention and at least oneother antibody which binds another tumor antigen associated with theCln101-expressing tumor cells. The cocktail may also comprise antibodiesthat are directed to other epitopes of Cln101. Preferably the otherantibodies do not interfere with the binding and or internalization ofthe antibodies of this invention.

The antibody therapeutic treatment method of the present invention mayinvolve the combined administration of an anti-Cln101 antibody (orantibodies) and one or more chemotherapeutic agents or growth inhibitoryagents, including co-administration of cocktails of differentchemotherapeutic agents. Chemotherapeutic agents include, e.g.,estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil,melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (such aspaclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparationand dosing schedules for such chemotherapeutic agents may be usedaccording to manufacturers' instructions or as determined empirically bythe skilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service Ed., M. C.Perry, Williams & Wilkins, Baltimore, Md. (1992).

The antibody may be combined with an anti-hormonal compound; e.g., ananti-estrogen compound such as tamoxifen; an anti-progesterone such asonapristone (see, EP 616 812); or an anti-androgen such as flutamide, indosages known for such molecules. Where the cancer to be treated isandrogen independent cancer, the patient may previously have beensubjected to anti-androgen therapy and, after the cancer becomesandrogen independent, the anti-Cln101 antibody (and optionally otheragents as described herein) may be administered to the patient.

Sometimes, it may be beneficial to also co-administer a cardioprotectant(to prevent or reduce myocardial dysfunction associated with thetherapy) or one or more cytokines to the patient. In addition to theabove therapeutic regimes, the patient may be subjected to surgicalremoval of cancer cells and/or radiation therapy, before, simultaneouslywith, or post antibody therapy. Suitable dosages for any of the aboveco-administered agents are those presently used and may be lowered dueto the combined action (synergy) of the agent and anti-Cln101 antibody.

For the prevention or treatment of disease, the dosage and mode ofadministration will be chosen by the physician according to knowncriteria. The appropriate dosage of antibody will depend on the type ofdisease to be treated, as defined above, the severity and course of thedisease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Preferably, the antibody isadministered by intravenous infusion or by subcutaneous injections.Depending on the type and severity of the disease, about 1 pg/kg toabout 50 mg/kg body weight (e.g. about 0.1-15 mg/kg/dose) of antibodycan be an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. A dosing regimen can comprise administering aninitial loading dose of about 4 mg/kg, followed by a weekly maintenancedose of about 2 mg/kg of the anti-Cln101 antibody. However, other dosageregimens may be useful. A typical daily dosage might range from about 1pg/kg to 100 mg/kg or more, depending on the factors mentioned above.For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. The progress of this therapy can be readilymonitored by conventional methods and assays and based on criteria knownto the physician or other persons of skill in the art.

Aside from administration of the antibody protein to the patient, thepresent application contemplates administration of the antibody by genetherapy. Such administration of a nucleic acid molecule encoding theantibody is encompassed by the expression “administering atherapeutically effective amount of an antibody”. See, for example, WO96/07321 published Mar. 14, 1996 concerning the use of gene therapy togenerate intracellular antibodies.

There are two major approaches to introducing the nucleic acid molecule(optionally contained in a vector) into the patient's cells; in vivo andex vivo. For in vivo delivery the nucleic acid molecule is injecteddirectly into the patient, usually at the site where the antibody isrequired. For ex vivo treatment, the patient's cells are removed, thenucleic acid molecule is introduced into these isolated cells and themodified cells are administered to the patient either directly or, forexample, encapsulated within porous membranes which are implanted intothe patient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187). Thereare a variety of techniques available for introducing nucleic acidmolecules into viable cells. The techniques vary depending upon whetherthe nucleic acid is transferred into cultured cells in vitro, or in vivoin the cells of the intended host. Techniques suitable for the transferof nucleic acid into mammalian cells in vitro include the use ofliposomes, electroporation, microinjection, cell fusion, DEAE-dextran,the calcium phosphate precipitation method, etc. A commonly used vectorfor ex vivo delivery of the gene is a retroviral vector.

The currently preferred in vivo nucleic acid molecule transfertechniques include transfection with viral vectors (such as adenovirus,Herpes simplex I virus, or adeno-associated virus) and lipid-basedsystems (useful lipids for lipid-mediated transfer of the gene areDOTMA, DOPE and DC-Cho1, for example). For review of the currently knowngene marking and gene therapy protocols see Anderson et at., Science256:808-813 (1992). See also WO 93/25673 and the references citedtherein.

Articles of Manufacture and Kits

The invention also relates to an article of manufacture containingmaterials useful for the detection for Cln101 overexpressing cellsand/or the treatment of Cln101 expressing cancer, in particular prostateor ovarian cancer. The article of manufacture comprises a container anda composition contained therein comprising an antibody of thisinvention. The composition may further comprise a carrier. The articleof manufacture may also comprise a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition which is effective for detecting Cln101 expressing cellsand/or treating a cancer condition and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is an anti-Cln101 antibody of theinvention. The label or package insert indicates that the composition isused for detecting Cln101 expressing cells and/or for treating prostateor ovarian cancer, or more specifically ovarian serous adenocarcinoma,breast infiltrating ductal carcinoma, prostate adenocarcinoma, renalcell carcinomas, colorectal adenocarcinomas, lung adenocarcinomas, lungsquamous cell carcinomas, and pleural mesothelioma, in a patient in needthereof. The breast cancer may be HER-2 negative or positive breastcancer. The cancers encompass metastatic cancers of any of thepreceding, e.g., prostate or ovarian cancer metastases. The label orpackage insert may further comprise instructions for administering theantibody composition to a cancer patient. Additionally, the article ofmanufacture may further comprise a second container comprising asubstance which detects the antibody of this invention, e.g., a secondantibody which binds to the antibodies of this invention. The substancemay be labeled with a detectable label such as those disclosed herein.The second container may contain e.g., a pharmaceutically-acceptablebuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution. Thearticle of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., forCln101 cell killing assays, for purification or immunoprecipitation ofCln101 from cells or for detecting the presence of Cln101 in a serumsample or other bodily fluids or detecting the presence ofCln101-expressing cells in a cell sample. For isolation and purificationof Cln101, the kit can contain an anti-Cln101 antibody coupled to asolid support, e.g., a tissue culture plate or beads (e.g., sepharosebeads). Kits can be provided which contain the antibodies for detectionand quantitation of Cln101 in vitro, e.g. in an ELISA or a Western blot.As with the article of manufacture, the kit comprises a container and acomposition contained therein comprising an antibody of this invention.The kit may further comprise a label or package insert on or associatedwith the container. The kits may comprise additional components, e.g.,diluents and buffers, substances which bind to the antibodies of thisinvention, e.g., a second antibody which may comprise a label such asthose disclosed herein, e.g., a radiolabel, fluorescent label, orenzyme, or the kit may also comprise control antibodies. The additionalcomponents may be within separate containers within the kit. The labelor package insert may provide a description of the composition as wellas instructions for the intended in vitro or diagnostic use.

EXAMPLES Example 1 Production and Isolation of Monoclonal AntibodyProducing Hybridomas

The following MAb/hybridomas of the present invention are describedbelow: Cln101.A1.1, Cln101.A3.1, Cln101.A6.1, Cln101.A7.1, Cln101.A8.1,Cln101.A9.1, Cln101.A10.1, Cln101.A11.1, Cln101.A15.1, Cln101.A16,Cln101.A17.1, Cln101.A18.1, Cln101.A23.1, Cln101.A26.1, Cln101.A28.1,Cln101.A35.1, Cln101.A37.1, Cln101.A38.1, Cln101.A41.1, Cln101.A42.1,Cln101.A46.1, Cln101.A47.1, Cln101.C3, Cln101.C6, Cln101.C12,Cln101.C17, Cln101.C18, Cln101.C25, Cln101.C36, Cln101.C43 andCln101.C47.

If the MAb has been cloned, it will get the nomenclature “X.1,” e.g.,the first clone of A2 will be referred to as A2.1, the second clone ofA2 will be referred to as A2.2, etc. For the purposes of this invention,a reference to MAb A2 will include all clones, e.g., A2.1, A2.2, etc.

Immunogens and Antigens (Recombinant Proteins, HA & His Tags &Transfected Cells)

For immunization of mice and production of MAbs, two Cln101 recombinantproteins were generated. The first was a bacterial (E. coli) expressedsoluble Cln101 recombinant protein. SEQ ID NO:1MASRSMRLLLLLSCLAKTGVLGDIIMRPSCAPGWFYHKSNCYGYFRKLRNWSDAELECQSYGNGAHLASILSLKEASTIAEYISGYQRSQPIWIGLHDPQKRQQWQWIDGAMYLYRSWSGKSMGGNKHCAEMSSNNNFLTWSSNECNKRQ HFLCKYRP,.

A second Cln101 construct was cloned with a histidine tag immediatelydownstream of codon Ser161. For production of the C-series of MAbs thisprotein was cloned into a standard expression vector and expressed inmammalian cells using standard technology known to those of skill in theart. SEQ ID INO:2 MLQNSAVLLVLVISASATHEAEQDIIMRPSCAPGWFYHKSNCYGYFRKLRNWSDAELECQSYGNGAHLASILSLKEASTIAEYISGYQRSQPIWIGLHDPQKRQQWQWIDGAMYLYRSWSGKSMGGNKHCAEMSSNNNFLTWSSNECNKRQHFLCKYRPASHHHHHHHHHH,.Immunizations

For generation of the A series MAbs mice were immunized with bacterialexpressed soluble Cln101 recombinant protein, SEQ ID NO:1. Groups of 8BALB/c mice were immunized intradermally in both rear footpads. Allinjections were 25 uL per foot The first injection (day 1) of 10 ug ofantigen per mouse was in Dulbecco's phosphate buffered saline (DPBS)mixed in equal volume to volume ratio with Titermax gold adjuvant(Sigma, Saint Louis, Miss.). Subsequent injections of 10 ug of antigenper mouse occurred on days 5, 9, 12, 16, 19, 23, 26, 29, 30 andconsisted of antigen in 20 uL of DPBS plus 5 uL of Adju-phos adjuvant(Accurate Chemical & Scientific Corp., Westbury, N.Y.) per mouse. Thefinal boost injection on day 33 consisted of antigen diluted in DPBSalone. Fusion occurred on Day 37.

For generation of the C-series MAbs mice were immunized as above withsoluble mammalian expressed Cln101 recombinant protein, SEQ ID NO:2.

Hybridoma Fusions

Mice were sacrificed at the completion of the immunization protocol anddraining lymph node (popliteal) tissue was collected by steriledissection. Lymph node cells were dispersed by pressing through asterile sieve into DMEM and removing T-cells via anti-CD90 (Thy1.2)coated magnetic beads (Miltenyl Biotech, Baraisch-Gladbach, Germany).

These primary B-cell enriched lymph node cells were then immortalized byelectro-cell fusion (BTX, San Diego, Calif.) with the continuous myelomacell line P3x63Ag8.653 (Kearney, J. F. et al., J. Immunology 123:1548-1550, 1979). Successfully fused cells were selected by culturing instandard Hypoxanthine, Azaserine (HA) (Sigma) containing selectionmedium (DMEM/10% FBS). These fusion cultures were immediatelydistributed, 10 million cells per plate, into wells of 96 well cultureplates. Distributing the culture in 96 well culture plates, immediatelyfollowing fusion, facilitated selection of a larger diversity ofhybridoma clones producing single, specific antibodies. Supernatantsfrom wells were screened by ELISA, for reactivity against Cln101protein.

Monoclonal cultures, consisting of the genetically uniform progeny fromsingle cells, were established after the screening procedure above, bysorting of single viable cells into wells of two 96 well plates, usingflow cytometry (Coulter Elite). The resulting murine B-cell hybridomacultures were expanded using standard tissue culture techniques.Selected hybridomas were cryopreserved in fetal bovine serum (FBS) with10% DMSO and stored in Liquid Nitrogen at −196° C. to assure maintenanceof viable clone cultures.

Screening & Selection of Antibody Producing Hybridomas

Hybridoma cell lines were selected for production of Cln101 specificantibody by enzyme linked solid phase immunoassay (ELISA). Cln101 orPro104 proteins were nonspecifically adsorbed to wells of 96 wellpolystyrene EIA plates (VWR). Fifty μL of Cln101 or Pro104 protein at0.91 mg/mL in (DPBS) was incubated overnight at 4° C. in wells of 96well polystyrene EIA plates. Plates were washed twice with Tris bufferedsaline with 0.05% Tween 20, pH 7.4 (TBST). The plate wells were thenemptied and nonspecific binding capacity was blocked by completelyfilling the assay wells with TBST/0.5% bovine serum albumin (TBST/BSA)and incubating for 30 minutes at room temperature (RT). The plate wellswere then emptied, 50 μL of hybridoma culture medium samples was addedto the wells and incubated for 1 hour at RT. The wells were then washed3 times with (TBST). One hundred uL of alkaline phosphatase conjugatedgoat anti-mouse IgG (Fc) (Pierce Chemical Co., Rockford, Ill.), diluted1:5000 in TBST/BSA, was then added to each well and incubated for 1 hourat RT. The wells were then washed 3 times with TBST. One hundred uL ofalkaline phosphatase substrate para-nitrophenylphosphate (pNPP) (Sigma)at 1 mg/mL in 1 M Diethanolamine buffer pH 8.9 (Sigma) was then added toeach well and incubated for 20 min. at RT. Bound alkaline phosphataseactivity was indicated by the development of a visible yellow color. Theenzymatic reaction was quantitated by measuring the solution'sabsorbance at 405 nm wavelength. Cultures producing the highestabsorbance values are chosen for expansion and further evaluation.

Western Blots

Protein extracts for western blot analysis were prepared in cell lysisbuffer (1% NP40, 10 mM Sodium Phosphate pH 7.2, 150 mM Sodium Chloride)from Cln101 expressing cell lines. Proteins were separated byelectrophoresis on NuPAGE 4-12% Bis-Tris gels (Invitrogen LifeTechnologies, Carlsbad, Calif.) under denaturing conditions inNovex-XCell II Minicell gel apparatus (Invitrogen, Life Tech) andsubsequently transferred to PVDF membranes using an XCell II Blot Module(Invitrogen Life Technologies). Following the transfer of proteins, themembranes were blocked in 1% blocking reagent (Roche Diagnostic Corp.,Indianapolis, Ind.) and incubated overnight at 4° C. with purifiedprimary antibodies (Cln101 monoclonal antibodies: A46.1, A17.1, A9.1,A6.1 A26.1 and A38.1) and then with horseradish-peroxidase conjugatedgoat anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc.) andfinally visualized by chemiluminescence using an ECL advance westernblotting detection kit (Amersham Biosciences, Piscataway, N.J.).

The preferred antibodies were Anti Cln101 Mab A46.1, A17.1, A9.1, A6.1A26.1 and A38.1

ProteinChip SELDI Immunoassay for Cln101.

The ProteinChip SELDI mass spectrometry biosystem was obtained fromCiphergen Biosystems, Inc., 6611 Dumbarton Circle, Fremont, Calif.94555. Preactivated surface ProteinChip arrays PS20 (CiphergenBiosystems) have carbonyl diimidazole moieties that can react covalentlywith their amine groups. 0.4 ug of purified MAB A16B recognizing theCln101 was applied on each spot of a PS20 chip and incubated for 16 h at4° C. or 1 h at 37° C. in a humidity chamber. An irrelevant MAB was usedas a control. Residual active sites were then blocked by incubating thearray in a 15-ml conical tube with 8 ml of 1 M ethanolamine, for 30 min,on a shaking platform. After three washes of 5 min each with 0.05%Triton X-100 in PBS (pH 7.4) followed by 3 washes for 5 min with PBS (pH7.4) in a 15-ml conical tube on a shaking platform, the chip wasincubated for 4 h in a humidity chamber with 100 μl of normal and breastserum sample, 100 ul of A549, HT29, PC-3 cell line medium at 100 ng/mlusing bioprocessor (Ciphergen Biosystems). The chip was washed aspreviously with 0.05% Triton X-100 in PBS (pH 7.4) and PBS (pH 7.4).Sinapinic acid was applied on each spot and mass spectrometry analysiswas performed in a PBS-II mass reader (Ciphergen Biosystems). Spectrawere collected using an average 80 nitrogen laser shots with a laserintensity of 280 and 290 and a detector sensitivity of 10. Spectrumanalysis was performed using the ProteinChip software version 2.1(Ciphergen Biosystems). The result of ProteinChip SELDI Immunoassay forCln101 showed Mab 16B could capture a unique protein from HT29 mediumabout 16 kd, well other control antibodies could not. The size ofcaptured protein is very close to the full length of Cln101 17 Kd,suggested that the captured protein in serum is Cln101. The capturing ofCln101 from serum was not successful.

Mab/Mab ELISA. A sequential sandwich ELISA assay was used to measurelevels of Cln101 in serum. Assay consisted of monoclonal Ab capture,monoclonal Ab-biotin and Alk Phos detector. High binding polystyrenestrip-wells were obtained from Corning Life Sciences (MA). The plateswere coated overnight at 4oC with 0.5 μg/well of anti-Cln101 MAb. Thecoating solution was then aspirated off and blocked with 300 μl/well ofSuperblock-TBS (Pierce Biotechnology, Illinois) for 1 hour at roomtemperature under shaking conditions. The blocking solution was thenaspirated off and 20 μl of Cln101 Standards diluted in 100% heatinactivated fetal bovine serum (Hyclone, Utah) at concentrations of6.25, 1.25, 0.5, 0.25, 0.1 and 0 ng/ml were then added to the wells. 20μl of serum was also added to the other wells. 100 μl of Assay buffercontaining 1% mouse serum, 10% FBS, and 1×TBS (Teknova, California) wasthen added to the 20 μl of samples and standards, and incubated at RT,shaking for 1 hour. The plate was washed four times with 360 μl1×TBS+0.05% Tween-20 (Teknova, California). Biotinylated Mab anti-Cln101was diluted as 0.75 ug/ml in 1×TBS pH 7.4 (Teknova, California) in AssayBuffer and 100 μl was added to each well and then incubated for 1 hourat room temperature while shaking. The plate was washed four times with360 μl 1×TBS+0.05% Tween-20 (Teknova, California). 100 μl of AlkalinePhosphatase conjugated Streptavidin, 1:2000 diluted in 1×TBS pH 7.4(Jackson ImmunoResearch Laboratories, Pennsylvania) was added to eachwell and incubated for 30 minutes at RT while shaking. The plate waswashed four times with 360 μl l×TBS+0.05% Tween-20 (Teknova,California). The plate was developed using pNPP substrate in 1×DEAbuffer (Pierce Biotechnology, Illinois) for 20 minutes at RT whileshaking. The reaction was stopped using 100 μl/well 1N NaOH, and read at405 nm using a Spectramax 190 plate reader (Molecular Devices,California).

Example 2 Monoclonal Sandwich ELISA Detection of Cln101

High binding polystyrene plates (Corning Life Sciences (MA)) were coatedovernight at 4° C. with 0.8 μg/well of anti-Cln101 MAb. The coatingsolution was aspirated off and free binding sites were blocked with 300μl/well Superblock-TBS (Pierce Biotechnology, Illinois) plus 100% calfserum for 1 hour at room temperature (RT). After washing 4× withTBS+0.1% Tween20, 50 μl of Assay Buffer (TBS, 1% BSA, 1% mouse Serum, 1%Calf Serum, 0.1% Tween20) was added to each well and then 50 μl ofantigen was added for 90 minutes incubation. For the checkerboardexperiment, each pair was tested on 50 ng/ml and 0 ng/ml of recombinantCln101. For each sandwich ELISA, standards of 10, 2.5, 0.5, 0.25, 0.1and 0 ng/ml Cln101 were run in parallel with the test samples. Standardsand test samples were diluted in Assay Buffer. For the detection, 100 μlof biotinylated MAb (1 μg/ml) were added to each well and incubated for1 hour at room temperature, while shaking. After washing, 100 μl ofhorseradish peroxidase conjugated streptavidin (1 mg/ml, JacksonImmunoResearch Laboratories, Pennsylvania) at a 1:20.000 dilution wasadded to each well and incubated for 30 minutes at RT while shaking.After washing, the plate was then developed using DAKO TMB Plussubstrate (DAKO, Denmark) for 30 minutes at RT. The reaction was stoppedusing 100 μl/well 1N HCL, and the plates were read at 450 nm using aSpectramax 190 plate reader (Molecular Devices, California).

The results of the checkerboard ELISA for the anti-Cln101 A-series MAbsusing bacterial recombinant Cln101 are shown in the table below.Identification of anti-Cln101 Antibody A-series Pairs Coating DetectingMAb MAb A1.1 A3.1 A6.1 A7.1 A8.1 A9.1 A10.1 A11.1 A12.1 A15.1 A16.1A17.1 A1.1 1 1 1 1 1 1 1 1 1 1 1 1 A3.1 1 1 1 1 1 1 1 1 1 1 1 1 A6.1 1 11 1 1 1 9 1 1 1 18 1 A7.1 1 1 1 1 1 1 9 1 1 1 16 1 A8.1 1 1 1 1 1 1 1 11 1 1 1 A9.1 1 1 1 1 1 1 21 1 1 1 1 1 A10.1 1 1 41 41 1 45 1 1 1 1 20 43A11.1 1 1 1 1 1 4 1 1 1 1 1 1 A12.1 1 1 1 1 1 1 1 1 1 1 1 1 A15.1 1 1 11 1 1 1 1 1 1 1 1 A16.1 1 1 35 19 1 1 3 1 1 1 1 1 A17.1 1 1 1 1 1 1 5 11 1 1 1 A18.1 0 1 1 1 1 1 1 1 1 1 1 1 A23.1 1 1 1 10 3 50 1 1 1 5 13 31A26.1 1 1 27 24 1 1 2 5 1 10 1 20 A28.1 1 1 1 1 1 1 1 1 1 1 1 1 A35.1 11 1 1 1 1 1 1 1 1 1 1 A37.1 1 1 9 1 1 1 1 1 1 1 1 14 A38.1 1 1 34 33 1 112 7 1 17 1 27 A41.1 1 1 1 1 1 1 1 1 1 1 1 1 A42.1 1 1 1 1 1 2 1 1 1 1 11 A46.1 1 1 1 1 1 40 1 1 1 2 27 32 A47.1 1 1 1 1 1 1 1 1 I 1 1 1 CoatingDetecting MAb MAb A18.1 A23.1 A26.1 A28.1 A35.1 A37.1 A38.1 A41.1 A42.1A46.1 A47.1 A1.1 1 1 1 1 1 1 1 1 1 1 1 A3.1 0 1 1 0 1 1 1 1 1 1 1 A6.1 13 33 1 1 1 33 1 1 1 1 A7.1 1 24 18 1 1 1 36 1 1 1 1 A8.1 1 20 1 1 1 1 281 1 2 1 A9.1 1 31 1 1 1 1 1 1 2 36 1 A10.1 1 1 28 1 1 1 47 1 1 1 1 A11.11 26 24 1 1 1 31 1 1 3 1 A12.1 1 1 1 1 1 1 1 1 1 1 1 A15.1 1 19 18 1 1 130 1 1 3 1 A16.1 1 30 1 1 1 1 1 1 1 32 1 A17.1 1 23 11 1 1 1 23 1 1 30 1A18.1 1 27 23 1 1 1 28 1 1 16 1 A23.1 32 1 22 1 1 1 41 1 45 1 1 A26.1 2722 1 1 1 1 1 1 29 28 1 A28.1 1 1 1 1 2 1 1 1 1 1 1 A35.1 1 1 1 1 1 1 1 11 1 1 A37.1 1 5 1 1 1 1 1 1 1 2 1 A38.1 25 27 1 1 1 1 1 1 28 30 1 A41.11 1 1 1 1 1 1 1 1 1 1 A42.1 1 28 23 1 1 1 30 1 1 27 1 A46.1 14 1 33 1 11 42 1 40 1 1 A47.1 1 1 1 1 1 1 1 1 1 1 1

The results of the checkerboard ELISA for the anti-Cln101 C-series MAbsusing mammalian recombinant Cln101 are shown in the table below.Identification of anti-Cln101 Antibody C-series Pairs detecting Abcoating Ab A46 A9 C3 C6 C12 C17 C18 C25 C36 C43 C47 control A46 2 52 465 33 12 52 47 53 52 43 1 A9 43 1 1 44 41 49 3 46 3 8 3 1 C3 46 1 1 50 4550 9 50 9 48 8 1 C6 3 54 45 2 3 4 49 1 52 1 44 1 C12 5 2 12 2 1 2 9 1 171 11 1 C17 23 37 20 1 1 6 44 1 33 1 43 1 C18 47 1 2 44 51 45 16 44 16 4012 1 C25 49 45 44 1 1 2 45 1 48 1 43 1 C36 43 1 2 37 45 49 8 44 7 45 6 1C43 45 1 50 2 1 3 44 2 48 1 42 1 C47 42 1 2 45 47 50 13 50 16 50 14 1

The results of the checkerboard ELISA for the anti-Cln101 A-series andC-series MAbs using mammalian recombinant Cln101 are shown in the tablebelow. Identification of anti-Cln101 Antibody A and C-series PairsDetecting Ab Coating Ab A6 A9 A10 A26 A38 A42 A46 C6 C17 C25 C36 controlA6 1 1 13 10 13 1 1 1 1 1 2 1 A9 1 1 18 1 1 1 44 21 20 22 2 1 A10 5 21 12 5 1 1 1 1 1 48 1 A26 3 1 2 1 1 2 4 4 4 3 1 1 A38 4 1 3 1 1 5 11 9 9 92 1 A42 1 1 1 2 2 1 6 1 1 1 8 1 A46 1 51 1 10 23 16 1 1 1 13 50 1 C6 119 1 6 17 1 1 1 1 1 48 1 C17 1 9 1 4 18 1 1 1 1 1 48 1 C25 1 5 1 2 8 1 61 1 1 51 1 C36 1 1 24 1 1 20 51 52 48 49 5 1 control 1 1 1 1 1 1 1 1 1 11 1

For the checkerboard ELISA, all possible combination of antibodies, weretested for efficiency as coating or detecting reagents. The pair A9.1and A46.1 demonstrated an excellent signal/noise ratio and were furtherevaluated in sandwich ELISA assays to analyze the efficiency ofdetection of endogenous Cln101 in lysates from cancer cell lines andbody fluids. Additional antibody pairs for detecting Cln101 includeA10/C36, A46/C25, A46/C36, C6/A9, C6/C36, C36/A46, C36/C17, A9/C17 andA9/C6. Binding results are graphically represented in FIG. 1.

Example 3 Cln101 Assays

Human Serum Samples

The human cancer serum samples in primary #1, primary #2 and secondarypanel #1 were obtained from IMPATH-BCP, Inc. (Los Angeles, Calif.) andDiagnostic Support Service, Inc. (West Barnstable, Mass.). The humannormal serum samples were obtained from ProMedDx, LLC. (Norton, Mass.).The human disease serum samples, primary panel #3 (cancer serum), colon,prostate, lung and breast extended panels (cancer and benign serumsamples) and all the samples in legacy panel (cancer and normal serumsamples) were obtained from Diagnostic Support Service, Inc., (WestBarnstable, Mass.). Additional ovarian cancer samples were obtained fromDIAGNOSTIC ONCOLOGY CRO, Inc. (DOCRO) (Seymour, Conn.). The ELISA assayswere done in primary panel#1, primary panel #2, primary panel #3, benignpanel, ProMedDx normal panel, bioavailability panel, colon and prostateextended panels and DOCRO ovarian panel. The primary #1 and #2 panel has23 cancer serum samples for breast, Colon, Lung, Ovarian and prostatecancer each, plus 45 normal serums. The 25 cancer serum samples in eachtype covered different age (from 40 to 70) and mixed stage (stage 1 to4). All the normal serum samples were from ProMedDx, they covered a widerange of age (from 20 to 80) and both genders. The benign panels covered16 disease, they are: Renal; Cystitis; Hypertension; BPH; HepaticToxicity; Prostatitis; Cirrhosis; Colon polyps; Diabetes; UlcerativeColitis; Crohns; Nephrolithaisis; Polycystic; Glomerulonephritis;Pancreatitis; Diverticulitis. The bioavailability panel consists of 49individuals with 6 to 9 multiple draws in one month of period time. Thecolon extended panel has 50 cancer serum samples and 200 benign serumsamples (50 of each disease: Ulcerative Colitis; Crohns; Colon polyps;Diverticulitis). The prostate extended panel has 200 cancer serumsamples and 100 benign serum samples (50 BPH; 50 Prostatitis). The DOCROpanel has 57 ovarian cancer serum samples and 24 Endometriosis serumsamples.

FIGS. 2-8 show Cln101 in the all cancer panel, the prostate cancerpanel, and the prostate cancer panel with benign diseases. FIG. 9 showsPSA in the prostate cancer panel with benign diseases. FIG. 10 showsCln101 in ovarian cancer (DOCRO panel). For determination of PSA levels,the serum samples were tested with PSA ELISA kit from Biocheck Inc(Burlingame, Calif.) following the manufacturer's protocol.

Example 4 ROC Analysis of Cln101 and PSA

The ability of a test to discriminate diseased cases from normal casesis evaluated using Receiver Operating Characteristic (ROC) curveanalysis (Metz, 1978; Zweig & Campbell, 1993). ROC curves can also beused to compare the diagnostic performance of two or more laboratory ordiagnostic tests (Griner et al., 1981).

ROC curve is generated by plotting sensitivity against specificity foreach value. From the plot, the area under the curve (AUC) can bedetermined. The value for the area under the ROC curve (AUC) can beinterpreted as follows: an area of 0.84, for example, means that arandomly selected positive result has a test value larger than that fora randomly chosen negative result 84% of the time (Zweig & Campbell,1993). When the variable under study can not distinguish between the tworesult groups, i.e. where there is no difference between the twodistributions, the area will be equal to 0.5 (the ROC curve willcoincide with the diagonal). When there is a perfect separation of thevalues of the two groups, i.e. there no overlapping of thedistributions, the area under the ROC curve equals 1 (the ROC curve willreach the upper left corner of the plot).

The 95% confidence interval for the area can be used to test thehypothesis that the theoretical area is 0.5. If the confidence intervaldoes not include the 0.5 value, then there is evidence that thelaboratory test does have an ability to distinguish between the twogroups (Hanley & McNeil, 1982; Zweig & Campbell, 1993). ROC Analysis forProstate Cancer (normal vs. cancer) analysis for individual markers fordifferentiation of normal males (n = 121) from males with prostatecancer (n = 138). Statistic Cln101 PSA AUC (95% CI) 0.933 0.801(0.895-0.960) (0.747-0.848) Cutoff for best combination 0.306 3.7  ofSens/Spec. Sens./Spec. at best cutoff 89%/92% 82%/67% Sens. (@ 90%Spec.) (Cutoff) 90% (0.291) 34% (9.9)

FIG. 11 shows the area under the curve for Cln101 and PSA (normal vs.cancer). ROC Analysis for Prostate Cancer Synergistic effects (normalvs. cancer) Logistic regression results for differentiation of normalmales (n = 121) from males with prostate cancer (n = 138) - Synergisticeffects Statistic Cln101 Cln101 + PSA PSA ROC AUC 0.933 0.940  0.801Wald p-value <0.0001 Cln101: <0.0001 <0.0001 PSA: 0.002 Odds Ratio (OR)1.2E−13 Cln101: 2.2E−12 8.3E−12 PSA: 5.7E−8: R2  0.5093 0.5523  0.1843Sens. (@ 90% Spec.) 90% 85% 34%

ROC Analysis for Prostate Cancer (normals + benigns vs. cancer) Analysisfor individual markers for differentiation of normal males and maleswith benign conditions (n = 268) from males with prostate cancer (n =138) Statistic Cln101 PSA AUC (95% CI) 0.741 0.762 (0.695-0.783)(0.718-0.803) Cutoff for best combination 0.35  3.7  of Sens/Spec.Sens./Spec. at best cutoff 87%/50% 82%/60% Sens. (@ 90% Spec.) (Cutoff)36% (1.08) 31% (10.8)

FIG. 12 shows the area under the curve for Cln101 and PSA(normals+benigns vs. cancer). ROC Analysis for Prostate CancerSynergistic effects (normals + benigns vs. cancer) Logistic regressionfor differentiation of normal males and males with benign conditions (n= 268) from males with prostate cancer (n = 138) - Synerqistic effectsStatistic Cln101 Cln101 + PSA PSA ROC AUC 0.741  0.800  0.762 Waldp-value <0.0001  Cln101: <0.0001 <0.0001 PSA: <0.0001 Odds Ratio (OR)0.0011 Cln101: 0.004 1.4E−9 PSA: 1.5E−8 R2 0.1251 0.2374  0.1611 Sens.(@ 90% Spec.) 36% 44% 31%

Number of Serum Samples in PSA “Grey Zone” PSA PSA PSA Samples 2-4 ng/mL4-10 ng/mL 2-10 ng/mL Normal 47 44 91 (n = 121) Prostate Cancer 20 52 72(n = 138) Prostate Benign 40 40 80 (n = 147)

ROC Analysis for Prostate Cancer Synergistic effects (normal vs. cancer,4-10 ng/mL PSA) Logistic regression results for differentiation ofnormal males (n=44) from males with prostate cancer (n=52) Synergisticeffects in the diagnostic “grey zone” sub-population of patients withPSA 4.0-10.0 ng/ml Statistic Cln101 Cln101 + PSA PSA ROC AUC 0.937 0.9500.526 Wald p-value <0.0001 Cln101: <0.0001 0.6997 PSA: 0.0700 Odds Ratio(OR) 4.7E−13 Cln101: 2.6E−15 0.736 PSA: 11.8 R2  0.5252 0.5617 0.0014Sens. (@ 90% Spec.) 88% 92% 6%

ROC Analysis for Prostate Cancer Synergistic effects (norm/ben vs.cancer, 4-10 ng/mL PSA) Logistic regression results for differentiationof normal males and males with benign conditions (n=84) from males withprostate cancer (n=52) Synergistic effects in the diagnostic “grey zone”sub-population of patients with PSA 4.0-10.0 ng/ml Statistic Cln101Cln101 + PSA PSA ROC AUC 0.657 0.657 0.522 Wald p-value 0.0041 Cln101:0.0044 0.6634 PSA: 0.9937 Odds Ratio (OR) 0.041 Cln101: 0.041 0.769 PSA:0.995 R2 0.0547 0.0547 0.0011 Sens. (@ 90% Spec.) 25% 25% 5%

ROC Analysis for Prostate Cancer Synergistic effects (normal vs. cancer,2-10 ng/mL PSA) Logistic regression results for differentiation ofnormal males (n=91) from males with prostate cancer (n=72) Synergisticeffects in the diagnostic “grey zone” sub-population of patients withPSA 2.0-10.0 ng/ml Statistic Cln101 Cln101 + PSA PSA ROC AUC 0.9200.921  0.640 Wald p-value <0.0001 Cln101: <0.0001 0.0105 PSA: 0.3272Odds Ratio (OR) 5.6E−10 Cln101: 1.2E−9 0.213 PSA: 0.440 R2  0.46130.4667 0.0390 Sens. (@ 90% Spec.) 88% 81% 9%

ROC Analysis for Prostate Cancer Synergistic effects (norm/ben vs.cancer, 2-10 ng/mL PSA) Logistic regression for differentiation ofnormal males and males with benign conditions (n=171) from males withprostate cancer (n=72) Synergistic effects in the diagnostic “grey zone”sub-population of patients with PSA 2.0-10.0 ng/ml Statistic Clnl01Cln101 + PSA PSA ROC AUC 0.694 0.714  0.627 Wald p-value <0.0001 Cln101:<0.0001 0.0027 PSA: 0.0123 Odds Ratio (OR) 0.019 Cln101: 0.024 0.245PSA: 0.292 R2 0.0838 0.1063 0.0328 Sens. (@ 90% Spec.) 26% 28% 9%

ROC Analysis for Prostate Cancer Synergistic effects (normal vs. cancer,24 ng/mL PSA) Logistic regression results for differentiation of normalmales (n=47) from males with prostate cancer (n=20) Synergistic effectsin the diagnostic “grey zone” sub-population of patients with PSA2.0-4.0 ng/ml Statistic Cln101 Cln101 + PSA PSA ROC AUC 0.905  0.921 0.575 Wald p-value 0.0007 Cln101: 0.0010 0.4335 PSA: 0.1778 Odds Ratio(OR) 8.3E−7 Cln101: 1.4E−7 0.47 PSA: 0.14 R2 0.4137 0.4448 0.0095 Sens.(@ 90% Spec.) 62% 62% 24%

ROC Analysis for Prostate Cancer Synergistic effects (norm/ben vs.cancer, 2-4 ng/mL PSA) Logistic regression for differentiation of normalmales and males with benign conditions (n=87) from males with prostatecancer (n=20) Synergistic effects in the diagnostic “grey zone”sub-population of patients with PSA 2.0-4.0 ng/ml Statistic Cln101Cln101 + PSA PSA ROC AUC 0.733 0.733  0.500 Wald p-value 0.0024 Cln101:0.0023 0.9739 PSA: 0.8370 Odds Ratio (OR) 0.008 Cln101: 0.008 1.03 PSA:1.21 R2 0.1368 0.1373 0.000 Sens. (@ 90% Spec.) 57% 52% ˜2%

Example 5 ROC Analysis of Cln101 and CA125

The ability of a test to discriminate diseased cases from normal casesis evaluated using Receiver Operating Characteristic (ROC) curveanalysis (Metz, 1978; Zweig & Campbell, 1993). ROC curves can also beused to compare the diagnostic performance of two or more laboratory ordiagnostic tests (Griner et al., 1981).

ROC curve is generated by plotting sensitivity against specificity foreach value. From the plot, the area under the curve (AUC) can bedetermined. The value for the area under the ROC curve (AUC) can beinterpreted as follows: an area of 0.84, for example, means that arandomly selected positive result has a test value larger than that fora randomly chosen negative result 84% of the time (Zweig & Campbell,1993). When the variable under study can not distinguish between the tworesult groups, i.e. where there is no difference between the twodistributions, the area will be equal to 0.5 (the ROC curve willcoincide with the diagonal). When there is a perfect separation of thevalues of the two groups, i.e. there no overlapping of thedistributions, the area under the ROC curve equals 1 (the ROC curve willreach the upper left corner of the plot).

The 95% confidence interval for the area can be used to test thehypothesis that the theoretical area is 0.5. If the confidence intervaldoes not include the 0.5 value, then there is evidence that thelaboratory test does have an ability to distinguish between the twogroups (Hanley & McNeil, 1982; Zweig & Campbell, 1993). ROC Analysis forOvarian Cancer (normal + benign disease vs. cancer) analysis forindividual markers for differentiation of normal (n = 31) or benigndisease (endometriosis n = 24) patients from patients with ovariancancer (n = 57). Statistic Cln101 CA125 ROC AUC 0.947 0.772 Sens. @ 90%Spec. 88% 67% Spec. @ 90% Sens. 87%  4%

ROC Analysis for Ovarian Cancer synergistic effects (normal + benigndisease vs. cancer) analysis for individual markers for differentiationof normal (n = 31) or benign disease (endometriosis n = 24) patientsfrom patients with ovarian cancer (n = 57). Statistic Cln101 CA125CA125 + Cln101 ROC AUC 0.947 0.772 0.969 Sens. @ 90% Spec. 88% 67% 93%Spec. @ 90% Sens. 87%  4% 95%

ROC Analysis for Stage 1 and 2 Ovarian Cancer (normal + benign diseasevs. cancer) analysis for individual markers for differentiation ofnormal (n = 31) or benign disease (endometriosis n = 24) patients fromnatients with stage 1 and 2 ovarian cancer (n = 28). Statistic Cln101CA125 ROC AUC 0.921 0.696 Sens. @ 90% Spec. 79% 61% Spec. @ 90% Sens.75%  4%

ROC Analysis for Stage 1 and 2 Ovarian Cancer synergistic effects(normal + benign disease vs. cancer) analysis for individual markers fordifferentiation of normal (n = 31) or benign disease (endometriosis n =24) patients from patients with staqe 1 and 2 ovarian cancer (n = 28).Statistic Cln101 CA125 CA125 + Cln101 ROC AUC 0.947 0.772 0.958 Sens. @90% Spec. 88% 67% 89% Spec. @ 90% Sens. 87%  4% 85%

Cln101 showed efficacy in detecting ovarian cancer in samples whereCA125 levels were not indicative of ovarian cancer. In addition todetecting ovarian cancer with CA125 levels below 30 U/ml, Cln101 isuseful in detecting ovarian cancer where CA125 values are between 30 and40 U/ml or between 30-35 U/ml. ROC Analysis for CA125 negative (<30U/mL) Ovarian Cancer (normal + benign disease vs. cancer) analysis forindividual markers for differentiation of normal (n = 31) or benigndisease (endometriosis n = 16) patients from patients with CA125negative (<30 U/mL) Ovarian Cancer (n = 19). Statistic Cln101 ROC AUC0.940 Sens. @ 90% Spec. 84% Spec. @ 90% Sens. 83%Results

ROC analysis results demonstrated Cln101 is an excellent marker fordetecting ovarian cancer compared to known marker CA125. High Cln101 AUCscores indicate Cln101 is useful for detecting Stage 1 & 2 ovariancancer alone or in combination with known marker CA125. Additionally,high Cln101 AUC scores indicate Cln101 is useful for detecting ovariancancer in patients where known markers fail. Specifically, Cln101detects ovarian cancer in patients with what is considered normal CA125levels, CA125 <30 U/ml. Furthermore, multivariate (Cln101+CA125)analysis may improve sensitivity and specificity for detecting ovariancancer.

In summary, Cln101 is useful for detecting early stage ovarian cancer,ovarian cancer in general and discriminating between ovarian cancer andbenign ovarian disease. Only 25% of all ovarian cancer is found instage 1. If ovarian cancer is found in stage 1 therapy (e.g. surgery) isvery effective and the 5-year survival rate is 90%.

Example 6 Deposits

Deposit of Cell Lines and DNA

Hybridoma cell lines were deposited with the American Type CultureCollection (ATCC) located at 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., and accorded accession numbers.

The following hybridoma cell lines were deposited with ATCC, Cln101.A9.1and Cln101.A46.1. The names of the deposited hybridoma cell lines abovemay be shortened for convenience of reference. E.g. A01.1 corresponds toCln101.A01.1. These hybridomas correspond to the clones (with their fullnames) deposited with the ATCC. The table below lists the hybridomaclone deposited with the ATCC, the accorded ATCC accession number, andthe date of deposit. ATCC deposits Hybridoma ATCC Accession No. DepositDate Cln101.A9.1 PTA-5877 Mar. 23, 2004 Cln101.A46.1 PTA-5876 Mar. 23,2004

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations there under (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of deposit. The organisms will be made available by ATCC underthe terms of the Budapest Treaty, and subject to an agreement betweendiaDexus, Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the cultures to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 3 7 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the cultureson deposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedstrains are not to be construed as a license to practice the inventionin contravention of the rights granted under the authority of anygovernment in accordance with its patent laws. The making of thesedeposits is by no means an admission that deposits are required toenable the invention.

REFERENCES

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1. A method for assessing risk of prostate cancer in a patient whichcomprises measuring levels of both Cln101 and Prostate Specific Antigen(PSA) in the patient, analyzing a risk associated with the level of PSAand a risk associated with the level of Cln101, and using the combinedrisks to assess the risk of prostate cancer in the patient. 2-7.(canceled)
 8. A method for assessing risk of ovarian cancer in a patientwhich comprises measuring levels of Cln101 in the patient to assess therisk of ovarian cancer in the patient.
 9. The method of claim 8 furthercomprising measuring levels of CA125 in the patient, analyzing a riskassociated with the level of CA125 and a risk associated with the levelof Cln101, and using the combined risks to assess the risk of ovariancancer in the patient. 10-19. (canceled)
 20. A kit for diagnosing apatient's susceptibility to prostate cancer or ovarian cancer comprisinga suitable assay for measuring Cln101 levels wherein the levels ofCln101 are determined.
 21. (canceled)
 22. The kit of claim 20 fordiagnosing a patient's susceptibility to ovarian cancer furthercomprising a suitable assay for measuring CA125 levels wherein thelevels of both CA125 and Cln101 are determined.
 23. (canceled)
 24. Anisolated Cln101 antibody that binds to mammalian Cln101 in vivo or invitro.
 25. The antibody of claim 24 which internalizes upon binding toCln101 on a mammalian cell in vivo.
 26. The antibody of claim 24 whichis a monoclonal antibody, an antibody fragment or a chimeric or ahumanized antibody. 27-28. (canceled)
 29. The antibody of claim 26 whichis produced by a hybridoma selected from the group consisting ofAmerican Type Culture Collection accession number PTA-5877 and PTA-5876.30. The antibody of claim 24, wherein the antibody competes for bindingto the same epitope as the epitope bound by the monoclonal antibodyproduced by a hybridoma selected from the group consisting of ATCCaccession number PTA-5877 and PTA-5876.
 31. The antibody of claim 24which is conjugated to a growth inhibitory agent or a cytotoxic agent.32-44. (canceled)
 45. A cell that produces the antibody of claim
 26. 46.The cell of claim 45, wherein the cell is selected from the groupconsisting of hybridoma cells deposited under American Type CultureCollection accession number PTA-5877 and PTA-5876.
 47. (canceled)
 48. Acomposition comprising the antibody of claim 24, and a carrier. 49-50.(canceled)
 51. The composition of claim 48, wherein the antibody is ahuman or humanized antibody and the carrier is a pharmaceutical carrier.52. (canceled)
 53. A method of killing a Cln101-expressing cancer cell,comprising contacting the cancer cell with the antibody of claim 24,thereby killing the cancer cell. 54-67. (canceled)
 68. The method ofclaim 53, wherein the antibody is conjugated to a cytotoxic agent. 69.(canceled)
 70. The method of claim 68, wherein the antibody isadministered in conjunction with at least one chemotherapeutic agent.71-73. (canceled)
 74. A method for determining if cells in a sampleexpress Cln101 comprising (a) contacting a sample of cells with anCln101 antibody of claim 24 under conditions suitable for specificbinding of the Cln101 antibody to Cln101 and (b) determining the levelof binding of the antibody to cells in the sample, or the level ofCln101 antibody internalization by cells in said sample, wherein Cln101antibody binding to cells in the sample or internalization of the Cln101antibody by cells in the sample indicate cells in the sample expressCln101.
 75. (canceled)
 76. The method of claim 74 wherein said sample ofcells is from a subject who has a cancer, is suspected of having acancer or who may have a predisposition for developing cancer. 77-83.(canceled)
 84. A method for detecting Cln101 overexpression in a subjectin need thereof comprising, (a) combining a bodily fluid sample of asubject with an Cln101 antibody of claim 24 under conditions suitablefor specific binding of the Cln101 antibody to Cln101 in said bodilyfluid sample (b) determining the level of Cln101 in the bodily fluidsample, (c) comparing the level of Cln101 determined in step b to thelevel of Cln101 in a control, wherein an increase in the level of Cln101in the bodily fluid sample from the subject as compared to the controlis indicative of Cln101 overexpression in the subject. 85-88. (canceled)89. A screening method for antibodies that bind to an epitope which isbound by an antibody of claim 26 comprising, (a) combining anCln101-containing sample with a test antibody and an antibody of claim26 to form a mixture, (b) determining the level of Cln101 antibody boundto Cln101 in the mixture and (c) comparing the level of Cln101 antibodybound in the mixture of step (a) to a control mixture, wherein the levelof Cln101 antibody binding to Cln101 in the mixture as compared to thecontrol is indicative of the test antibody's binding to an epitope thatis bound by the anti-Cln101 antibody of claim
 26. 90-94. (canceled) 95.The kit of claim 20 for diagnosing a patient's susceptibility toprostate cancer further comprising a suitable assay for measuringProstate Specific Antigen (PSA) levels wherein the levels of both PSAand Cln101 are determined.